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item2 Neo-resistant recombinant ESCs were analyzed for correct 3' and 5' targeting through hybridization by standard protocols, and we identified seven correctly targeted clones. Following ESC cell injection, several high-percentage chimera males were born. These were bred with Cre-expressing mice, and Cre-mediated excision of Alkbh5 exon 1 was tested in 26 agouti F1 pups. Two animals tested positive for the excised allele. These two mice, heterozygous for the constitutive allele (Alkbh5+/-), were analyzed by Southern blot analysis and used for further breeding to generate Alkbh5-/- mice.
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item10 Eight adult female C57BL/6 mice from the same pool of litters were housed at 22 ± 1 ℃ with 12-h light cycle and were mated with their male siblings. The female mice were fed with a low-fat diet supplemented with betaine from mating and throughout gestation and lactation. Litters were culled to five males per dam. Thirty offspring weaned at the age of day 21 and then were randomly assigned to three groups fed with either a low-fat diet (LF), high-fat diet (HF), or high-fat diet supplemented with betaine (HB) for 6 weeks.
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item12 HCT116, Y2KD-116 and con-116 cells were resuspended at 1 × 106 cells per 50 ul of PBS. Cells were injected into the exteriorized spleen after abdominal incision.
item13 A total of 1,000 breast cancer cells were injected into the mammary fat pad of 6-8-wk-old female NSG mice in a 1:1 suspension of Matrigel (BD Biosciences) in PBS solution. At 10 wk after injection, mice were examined for the presence of tumors, which were harvested for analysis.
item14 1,000 MDA-MB-231 subclone cells were injected into the mammary fat pad of randomly chosen 6-to-8 week-old female severe combined immunodeficiency (SCID) mice in a 1:1 suspension of Matrigel (BD Biosciences) in PBS (n = 7 mice per subclone).
item15 Male athymic BALB/c nude mice (5 weeks old) were used. Subcutaneous tumor growth assays, liver metastasis model, and tail vein injection model were performed as described.Metastases were detected using the IVIS@Lumina II system (Caliper Life Sciences, Hopkinton, MA) 10 minutes after intraperitoneal injection of 4.0 mg luciferin (Gold Biotech) in 50 uL of saline.
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item20 Sixteen 10-wk-old male C57BL/6 J mice [wild-type (WT) mice] and six 10-wk-old male obese C57BL/6Job/ob (OB) mice were purchased from Nanjing Biomedical Research Institute of Nanjing University. WT mice were randomly divided into 2 groups (8 mice/group): normal chow diet (ND) and high-fat diet (HFD) groups. The ND group was fed with a normal chow diet containing10% fat, 20% protein and 70% carbohydrate. The HFD group was fed a HFD containing 45% fat, 20% protein and 35% carbohydrate. OB mice were fed with ND. All mice were housed at 22 ± 1 ℃ under a 12-h light cycle with free access to water and diet during the experiment.
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item23 For the animal survival analysis, mice were intracranially injected with 10,000 GSCs and maintained until moribund or 80 days after injection. For the rescue studies, GSCs with ALKBH5 or FOXM1-AS shRNAs were co-transfected with a FOXM1, ALKBH5 wild-type or mutant expression construct.
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item26 Mixture of Cas9 mRNA (20 ng/uL) and two sgRNAs (5 ng/uL each) were injected into the cytoplasm and male pronucleus of the zygote, obtained by CBF1 mating.
item27 All mice described above were maintained on the C57BL/6J (B6) background. Mettl3- and Mettl14-floxed mice (Mettl3flox/flox and Mettl14flox/flox) were then bred with germ cell-specific expressed Cre mice including Vasa-Cre mouse line (Jackson Laboratory, Bar Harbor, Maine, USA) and Stra8-GFPCre mouse line for excising the loxP-flanked exon 4 and exon 2 to generate germ cell-specific Mettl3 and Mettl14KO mice, respectively. Germ cell-specific Mettl3 and Mettl14 double KO mice were obtained by crossing Mettl3flox/floxMettl14flox/flox with Mettl3flox/+Mettl14flox/+ or Mettl3flox/+Mettl14flox/flox carried germ cell-specific expressed Cre mice.
item28 All mice described above were maintained on the C57BL/6J (B6) background. Mettl3- and Mettl14-floxed mice (Mettl3flox/flox and Mettl14flox/flox) were then bred with germ cell-specific expressed Cre mice including Vasa-Cre mouse line (Jackson Laboratory, Bar Harbor, Maine, USA) and Stra8-GFPCre mouse line for excising the loxP-flanked exon 4 and exon 2 to generate germ cell-specific Mettl3 and Mettl14KO mice, respectively. Germ cell-specific Mettl3 and Mettl14 double KO mice were obtained by crossing Mettl3flox/floxMettl14flox/flox with Mettl3flox/+Mettl14flox/+ or Mettl3flox/+Mettl14flox/flox carried germ cell-specific expressed Cre mice.
item29 500,000 selected cells were injected via tail vein or retro-orbital route into female NSG (6-8 week old) recipient mice that had been sublethally irradiated with 475 cGy one day before transplantation.
item30 500,000 selected cells were injected via tail vein or retro-orbital route into female NSG (6-8 week old) recipient mice that had been sublethally irradiated with 475 cGy one day before transplantation.
item31 500,000 selected cells were injected via tail vein or retro-orbital route into female NSG (6-8 week old) recipient mice that had been sublethally irradiated with 475 cGy one day before transplantation.
item32 500,000 selected cells were injected via tail vein or retro-orbital route into female NSG (6-8 week old) recipient mice that had been sublethally irradiated with 475 cGy one day before transplantation.
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item36 For the subcutaneous implantation model, 2 × 106 METTL3 stable knockdown Huh-7 cells or METTL3 overexpression MHCC97L cells were injected subcutaneously into BABL/cAnN-nude mice. For orthotopic implantation, wild-type and METTL3 knockout Huh-7 cells were luciferase labelled, and 2 × 106 cells were then injected orthotopically into the left liver lobe of nude mice.
item37 For the subcutaneous implantation model, 2 × 106 METTL3 stable knockdown Huh-7 cells or METTL3 overexpression MHCC97L cells were injected subcutaneously into BABL/cAnN-nude mice. For orthotopic implantation, wild-type and METTL3 knockout Huh-7 cells were luciferase labelled, and 2 × 106 cells were then injected orthotopically into the left liver lobe of nude mice.
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item40 Performed an ex vivo genome wide CRISPR dropout screen (Screen 1) using Cas9-expressing mouse primary leukaemia cells driven by an MLL-AF9 fusion gene and a FLT3 internal tandem duplication.
item41 Performed an ex vivo genome wide CRISPR dropout screen (Screen 1) using Cas9-expressing mouse primary leukaemia cells driven by an MLL-AF9 fusion gene and a FLT3 internal tandem duplication.
item42 Cells (5×106 cells in 200 uL) were suspended with 100 uL PBS and 100 uL Matrigel Matrix, and injected subcutaneously into the left armpit of each mouse.
item43 Cells (5×106 cells in 200 uL) were suspended with 100 uL PBS and 100 uL Matrigel Matrix, and injected subcutaneously into the left armpit of each mouse.
item44 Cells (5×106 cells in 200 uL) were suspended with 100 uL PBS and 100 uL Matrigel Matrix, and injected subcutaneously into the left armpit of each mouse.
item45 Cells (5×106 cells in 200 uL) were suspended with 100 uL PBS and 100 uL Matrigel Matrix, and injected subcutaneously into the left armpit of each mouse.
item46 For R-2HG injection mouse models, sensitive (NOMO-1 and MA9.3ITD) or resistant (MA9.3RAS) cells were injected into NSGS or NRGS intravenously, and then R-2HG (6mg/kg body weight) or PBS were injected once daily through tail vein for 12 consecutive days starting from day 11 post xeno-transplantation.
item47 For R-2HG injection mouse models, sensitive (NOMO-1 and MA9.3ITD) or resistant (MA9.3RAS) cells were injected into NSGS or NRGS intravenously, and then R-2HG (6mg/kg body weight) or PBS were injected once daily through tail vein for 12 consecutive days starting from day 11 post xeno-transplantation.
item48 For R-2HG injection mouse models, sensitive (NOMO-1 and MA9.3ITD) or resistant (MA9.3RAS) cells were injected into NSGS or NRGS intravenously, and then R-2HG (6mg/kg body weight) or PBS were injected once daily through tail vein for 12 consecutive days starting from day 11 post xeno-transplantation.
item49 For R-2HG injection mouse models, sensitive (NOMO-1 and MA9.3ITD) or resistant (MA9.3RAS) cells were injected into NSGS or NRGS intravenously, and then R-2HG (6mg/kg body weight) or PBS were injected once daily through tail vein for 12 consecutive days starting from day 11 post xeno-transplantation.
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item51 Lin- HSPCs were purified from BM of wildtype mice and 0.1×106 cells were seeded in 2 mL OP9 medium onto the OP9 cells with the addition of 10 ng/mL mouse IL-3, 10 ng/mL human IL-6, 10 ng/mL mouse IL-7, 10 ng/mL mouse Flt-3L, and 50 ng/mL mouse stem cell factor (SCF).
item52 Lin- HSPCs were purified from BM of wildtype mice and 0.1×106 cells were seeded in 2 mL OP9 medium onto the OP9 cells with the addition of 10 ng/mL mouse IL-3, 10 ng/mL human IL-6, 10 ng/mL mouse IL-7, 10 ng/mL mouse Flt-3L, and 50 ng/mL mouse stem cell factor (SCF).
item53 Either SiHa cells or FTO overexpressed SiHa cells were injected subcutaneously into the right flanks of 4- to 6-week-old female athymic nude mice (CAS, Beijing, China). The mice were divided into four groups (N = 6). After transplantation, tumor size was measured by caliper every other day. Tumor volume was calculated using the formula: volume = (length × width2)/2. When the tumor volumes reached 50 mm3, the animals were treated with intraperitoneal injection of cisplatin (3 mg/kg) every other day for seven times and local irradiation of 8 Gy one time.
item54 Either SiHa cells or FTO overexpressed SiHa cells were injected subcutaneously into the right flanks of 4- to 6-week-old female athymic nude mice (CAS, Beijing, China). The mice were divided into four groups (N = 6). After transplantation, tumor size was measured by caliper every other day. Tumor volume was calculated using the formula: volume = (length × width2)/2. When the tumor volumes reached 50 mm3, the animals were treated with intraperitoneal injection of cisplatin (3 mg/kg) every other day for seven times and local irradiation of 8 Gy one time.
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item57 To screen for meiotic defects, spermatocyte squash preparation and immunostaining for SYCP3 and γH2AX (described below) were carried out using testes from pubertal G3 males that were ≥15 dpp or from adult G3 males. At these ages, spermatocytes in the relevant stages of meiotic prophase I are abundant in normal mice.
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item59 To build the mouse model for POI, cyclophosphamide (CTX) (Sigma, St. Louis, MO) was used a high-dose treatment (120 mg/kg, 2 weeks).
item60 To build the mouse model for POI, cyclophosphamide (CTX) (Sigma, St. Louis, MO) was used a high-dose treatment (120 mg/kg, 2 weeks).
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item88 After being fed with high-fat diet for 4 weeks, mice were given twice vena caudalis injection of control siRNA or Cidec siRNA (50 ug/mouse) mixed with liposome. Liposomes were prepared as described elsewhere.
item89 After being fed with high-fat diet for 4 weeks, mice were given twice vena caudalis injection of control siRNA or Cidec siRNA (50 ug/mouse) mixed with liposome. Liposomes were prepared as described elsewhere.
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item100 Mice were anesthetized after 24 h of fasting, and 5 U of human insulin (Humalin R; Eli Lilly) was injected into the inferior vena cava. After 5 min, the liver and hind limb muscles were dissected and immediately frozen in liquid nitrogen.
item101 Mice were anesthetized after 24 h of fasting, and 5 U of human insulin (Humalin R; Eli Lilly) was injected into the inferior vena cava. After 5 min, the liver and hind limb muscles were dissected and immediately frozen in liquid nitrogen.
item102 Mice were anesthetized after 24 h of fasting, and 5 U of human insulin (Humalin R; Eli Lilly) was injected into the inferior vena cava. After 5 min, the liver and hind limb muscles were dissected and immediately frozen in liquid nitrogen.
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item104 All pups were genotyped and two Mettl3flox/+ founder mice were obtained.
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item108 The animals were kept in standard laboratory polycarbonate cages in controlled ambient temperature at 23℃ ± 1℃ with relative humidity of 50% ± 10%, and a light-dark cycle of 12/12 h. The mice had free access to standardized pellet food and tap water. After 1-week of habituation, the animals were randomly divided into the following 4 groups: control group, 0.5 mg/l arsenite group, 5 mg/l arsenite group and 50 mg/l arsenite group (n = 16 per group). The mice were exposed to arsenite via drinking water for 6 months.
item109 We quantified m6A levels in RNA extracted from failing human (both ischemic and non-ischemic), pig and mouse (post-myocardial infarction ischemic) hearts and compared them to m6A in non-failing human and sham surgical controls respectively. We detected significantly elevated levels of m6A in both total and polyA+ RNA extracted from human, pig and mouse failing left ventricular (LV) explants compared to non-failing or sham.
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item111 For the rescue experiment, LSK cells were sorted from wt and Ythdf2 KO mouse BM and cultured overnight in StemSpan SFEM medium (Stem Cell Technologies) supplemented with 10 ug/mL heparin (Sigma), 0.5 × penicillin/streptomycin (Sigma), 10 ng/mL recombinant mouse (rm) stem cell factor (SCF), and 20 ng/mL Tpo 75 at 37 ℃ in a 5% CO2 5% O2 atmosphere.
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item116 Utilized three Cre transgenic mouse lines (Mx1-Cre, Ert-Cre and Vav-Cre) to cross with Ythdf2fl/fl mice to deplete the expression of YTHDF2 specifically in adult HSCs of mouse bone marrow (BM), respectively.
item117 4×106 HEC-1-A endometrial cancer cells (shCtrl, shMETTL3, wild-type, METTL14+/-, or METTL14+/- rescued with wild-type or mutant METTL14) were injected intraperitoneally into 5 week old female athymic nude mice (Foxn1nu, Harlan; n=10 per group).
item118 4×106 HEC-1-A endometrial cancer cells (shCtrl, shMETTL3, wild-type, METTL14+/-, or METTL14+/- rescued with wild-type or mutant METTL14) were injected intraperitoneally into 5 week old female athymic nude mice (Foxn1nu, Harlan; n=10 per group).
item119 4×106 HEC-1-A endometrial cancer cells (shCtrl, shMETTL3, wild-type, METTL14+/-, or METTL14+/- rescued with wild-type or mutant METTL14) were injected intraperitoneally into 5 week old female athymic nude mice (Foxn1nu, Harlan; n=10 per group).
item120 4×106 HEC-1-A endometrial cancer cells (shCtrl, shMETTL3, wild-type, METTL14+/-, or METTL14+/- rescued with wild-type or mutant METTL14) were injected intraperitoneally into 5 week old female athymic nude mice (Foxn1nu, Harlan; n=10 per group).
item121 2 × 106 tumor cells (OVCAR3-METTL3 and OVCAR3-Ctrl) or 1 × 106 tumor cells (SKOV3-shMETTL3-1, SKOV3-shMETTL3-2 and SKOV3-shNC) were suspended in 200 uL of RPMI 1640 complete culture medium with 25% Matrigel (BD Biosciences) and inoculated subcutaneously into the right flank of the nude mice.
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item126 For tumor xenograft studies, MDA-MB-231 cells transfected with scrambled-siRNA or METTL14-siRNA or ALKBH5-siRNA (2 × 106) were mixed with Matrigel and injected subcutaneously in the flank of 6-week-old female athymic nude mice.
item127 For tumor xenograft studies, MDA-MB-231 cells transfected with scrambled-siRNA or METTL14-siRNA or ALKBH5-siRNA (2 × 106) were mixed with Matrigel and injected subcutaneously in the flank of 6-week-old female athymic nude mice.
item128 HNF4a knockout (HNF4a-/-) mice were generated using CRISPR/Cas9 in C57BL/6 mice.
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item130 5 × 106 of HEP3B and SMMC7721 stable cells were resuspended in 0.1 ml of PBS and subcutaneously injected into the flank of mice.
item131 Mettl3fl/+ mice with C57BL6/J background were generated using CRISPR-Cas9 systems.
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item136 BALB/c-nu mice (4-6 weeks old, female) were purchased from Charles River Laboratories (Beijing, China), and SUNE1-vector-luciferase or SUNE1-ZNF750-luciferase cells (1 × 106) were subcutaneously injected into the dorsal or ventral flank.
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item138 Mettl3 fl/fl mice were crossed with mice expressing cre recombinase under the control of the cardiac-specific Myh7 promoter (Beta-myosin heavy chain [Beta-MHC]) to obtain heart-restricted deletion of Mettl3 (METTL3-cKO; cKO).
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item140 Five- to 6-week-old male athymic nude mice purchased by Charles River were used for the xenograft model. 769-P cells stably expressing Ctrl, FTO and FTO-mut were trypsinized and washed twice to thrice with standardized PBS, and then, 5 × 106 cells in 100 uL of PBS was subcutaneously injected into the flanks of the mice (five mice per group). Mice were monitored twice every week for tumour growth, and tumour diameters were measured using a caliper.
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item154 Positive ES cell clones were expanded and injected into C57BL/6J blastocysts to generate the chimeric offspring. The chimeric mice were mated with C57BL/6J mice to obtain the lnc-Dpf3 floxed heterozygous mouse (lnc-Dpf3fl/+). The lnc-Dpf3fl/+ mice were bred with C57BL/6J mice expressing the Cre recombinase under the control of the Itgax promoter (Itgax-cre mice; The Jackson Laboratory) to generate lnc-Dpf3 floxed heterozygous mouse with Itgax-cre allele (lnc-Dpf3fl/+Itgax-cre+). These mice were intercrossed to generate DC-specific lnc-Dpf3-deficient mice (lnc-Dpf3fl/flItgax-cre+) used for this study. Hif1afl/fl mice, a kindly gift from Dr. Ming Zhang (Renji Hospital, Medical College of Shanghai Jiaotong University, China) were crossed with Itgax-cre mice to generate DC-specific HIF-1-Alpha-deficient mice (Hif1afl/flItgax-cre+). All mice were maintained under specific pathogen-free conditions and used at 6-8 weeks of age.
item155 Two weeks after the stereotaxic surgery, all the animals were intraperitoneally injected with apomorphine at a dose of 0.5 mg/kg to induce the contralateral rotations. Ten minutes after the injection, a video was used to record the rotations of each rat for 20 min. Only those 6-OHDA induced rats showing robust contralateral turning (>7 turns/min) that were injected with 6-OHDA were used in subsequent experiments.
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item158 To cause I/R injury, mice were subjected to 30 min of LAD ischemia followed by 60 min of reperfusion.
item159 To cause I/R injury, mice were subjected to 30 min of LAD ischemia followed by 60 min of reperfusion.
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item168 Equal numbers of A549 cells expressing either control or shFTO were injected subcutaneously, within 30 min of harvesting, over the right and left flanks in male nu/nu mice between 4 and 6 weeks of age.
item169 Equal numbers of A549 cells expressing either control or shFTO were injected subcutaneously, within 30 min of harvesting, over the right and left flanks in male nu/nu mice between 4 and 6 weeks of age.
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item172 MDSCs were from C57BL/6 or C57BL/6 IL6 KO mice. B16 tumor cells were injected into wild-type (WT) and IL6 KO mice. PBS was used as a control.
item173 For the subcutaneous implantation model, 5 4-week-old female Balb/c mice were randomly grouped and injected with 1 × 106 shCtrl, shFTO or shFTO/shBNIP3 KD 4 T1 cells. For tumor metastasis mouse model, 5 4-week-old female Balb/c mice were randomly grouped and injected with 1 × 106 shCtrl, shFTO or shFTO/shBNIP3 KD 4 T1 cells via tail vein. For orthotopic xenograft mouse model, 5 4-week-old female NOD/SCID mice were randomly grouped.
item174 HPAF-II cells (2.0 × 106 cells/site) stably transfected with sh-EGFP or sh-UCA1 were subcutaneously injected into 4-week-old nude mice to generate xenografts. The tumor volume was measured every week after injection and calculated using the following formula: length × (width2)/2.
item175 HPAF-II cells (2.0 × 106 cells/site) stably transfected with sh-EGFP or sh-UCA1 were subcutaneously injected into 4-week-old nude mice to generate xenografts. The tumor volume was measured every week after injection and calculated using the following formula: length × (width2)/2.
item176 HCT-15 RP11 stable overexpression or control cells (2 × 106 per mouse) diluted in 100 uL normal medium + 100 uL Matrigel (BD Biosciences) were subcutaneously injected into immunodeficient mice to investigate tumour growth.
item177 HCT-15 RP11 stable overexpression or control cells (2 × 106 per mouse) diluted in 100 uL normal medium + 100 uL Matrigel (BD Biosciences) were subcutaneously injected into immunodeficient mice to investigate tumour growth.
item178 HCT-15 RP11 stable overexpression or control cells (2 × 106 per mouse) diluted in 100 uL normal medium + 100 uL Matrigel (BD Biosciences) were subcutaneously injected into immunodeficient mice to investigate tumour growth.
item179 HCT-15 RP11 stable overexpression or control cells (2 × 106 per mouse) diluted in 100 uL normal medium + 100 uL Matrigel (BD Biosciences) were subcutaneously injected into immunodeficient mice to investigate tumour growth.
item180 SKOV3 or A2780 cells were infected with the indicated lentiviral vectors and injected (5 × 106 cells/mouse in 200 uL volume) subcutaneously into the left armpit of 6-week-old BALB/c nude mice. After 21 days, the animals were sacrificed to confirm the presence of tumors and weigh the established tumors
item181 SKOV3 or A2780 cells were infected with the indicated lentiviral vectors and injected (5 × 106 cells/mouse in 200 uL volume) subcutaneously into the left armpit of 6-week-old BALB/c nude mice. After 21 days, the animals were sacrificed to confirm the presence of tumors and weigh the established tumors
item182 SKOV3 or A2780 cells were infected with the indicated lentiviral vectors and injected (5 × 106 cells/mouse in 200 uL volume) subcutaneously into the left armpit of 6-week-old BALB/c nude mice. After 21 days, the animals were sacrificed to confirm the presence of tumors and weigh the established tumors
item183 SKOV3 or A2780 cells were infected with the indicated lentiviral vectors and injected (5 × 106 cells/mouse in 200 uL volume) subcutaneously into the left armpit of 6-week-old BALB/c nude mice. After 21 days, the animals were sacrificed to confirm the presence of tumors and weigh the established tumors
item184 SKOV3 or A2780 cells were infected with the indicated lentiviral vectors and injected (5 × 106 cells/mouse in 200 uL volume) subcutaneously into the left armpit of 6-week-old BALB/c nude mice. After 21 days, the animals were sacrificed to confirm the presence of tumors and weigh the established tumors
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item186 Mice (five in each group) were injected subcutaneously with 0.1 ml of cell suspension containing 2 × 106 cells in the back flank.
item187 THP-1 cells transduced with CTL or KD lentiviruses were tail vein injected into non-irradiated 12 week-old female non-obese diabetic (NOD)/LtSz-severe combined immune-deficiency (SCID) IL-2Rγcnull (NSG) mice (1x106 cells per 200 uL per mouse).
item188 All animal experiments complied with Zhongshan School of Medicine Policy on Care and Use of Laboratory Animals. For subcutaneous transplanted model, sh-control and sh-METTL3 Huh7 cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 uL of PBS + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient female mice to investigate tumor growth.
item189 All animal experiments complied with Zhongshan School of Medicine Policy on Care and Use of Laboratory Animals. For subcutaneous transplanted model, sh-control and sh-METTL3 Huh7 cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 uL of PBS + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient female mice to investigate tumor growth.
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item195 Mice were anesthetized and shaved on both flanks and injected subcutaneously with 4 × 106 tumor cells in 200 uL of PBS.
item196 1 × 105 parental HT29-shNC cells, HT29-shYTHDF1 cells, HT29-shNC colonospheres, and HT29-shYTHDF1 colonospheres were inoculated subcutaneously into the left inguinal folds of the nude mice.
item197 1.5 × 106 TFK1 cells with or without FTO overexpression were injected subcutaneously into the left and right flanks of 6-week-old female athymic nude mice
item198 2 × 106 cells were suspended in 150 uL DMEM. The A431 cells (scrambled group and shRNA1 group) were injected subcutaneously into the flanks of nude mice.
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item200 The right knee joint of each OA mouse was injected with 1U of type VII collagenase over two consecutive days to obtain experimental OA joint, and the control mice received the equal volume of physiological saline.
item201 The right knee joint of each OA mouse was injected with 1U of type VII collagenase over two consecutive days to obtain experimental OA joint, and the control mice received the equal volume of physiological saline.
item202 About 1× 107 cells were injected subcutaneously into the axilla of the female athymic BALB/C nude mice (4-6 weeks old, 18-22 g, five mice per group).
item203 About 1× 107 cells were injected subcutaneously into the axilla of the female athymic BALB/C nude mice (4-6 weeks old, 18-22 g, five mice per group).
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item207 When the tumors reached a volume of 80-100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 ug/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody or isotype control IgG (250 ug/mouse) every other day after tumor cell inoculation.
item208 When the tumors reached a volume of 80-100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 ug/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody or isotype control IgG (250 ug/mouse) every other day after tumor cell inoculation.
item209 When the tumors reached a volume of 80-100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 ug/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody or isotype control IgG (250 ug/mouse) every other day after tumor cell inoculation.
item210 When the tumors reached a volume of 80-100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 ug/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody or isotype control IgG (250 ug/mouse) every other day after tumor cell inoculation.
item211 When the tumors reached a volume of 80-100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 ug/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody or isotype control IgG (250 ug/mouse) every other day after tumor cell inoculation.
item212 When the tumors reached a volume of 80-100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 ug/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody or isotype control IgG (250 ug/mouse) every other day after tumor cell inoculation.
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item215 The Metastatic Bone Tumor Model was established via injecting HOS cells into the right tibia of each animal.
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item230 Xenograft tumors were generated by injection (5.0 × 106 cells/injection) of shMTHFD2 786-O and CAKI-1 lines and their respective shControl cell lines (n = 6 per line, sample size calculated for 80% power to detect a twofold difference with 95% confidence) subcutaneously into 6-week-old male Nu/J mice.
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item233 For the tumor growth model, 1 × 106 HNE1-Scrambled or HNE1-shFAM2225A 2# cells were injected into the axilla of the mice, and the tumor size was measured every 3 days.
item234 For the tumor growth model, 1 × 106 HNE1-Scrambled or HNE1-shFAM2225A 2# cells were injected into the axilla of the mice, and the tumor size was measured every 3 days.
item235 Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO.
item236 Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO.
item237 Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO.
item238 Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO.
item239 .
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item246 .
item247 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item248 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item249 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item250 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item251 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item252 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item253 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item254 Hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were generated by crossing mice with TBG-Cre Tg mice. METTL3 flox (METTL3 fl/fl) and hepatocyte-specific METTL3 knockout mice (TBG-Cre, METTL3 fl/fl) were used for experiments.
item255 For the subcutaneous implantation model, 1 × 107 cells were subcutaneously implanted into 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice.
item256 For the subcutaneous implantation model, 1 × 107 cells were subcutaneously implanted into 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice.
item257 For the subcutaneous implantation model, 1 × 107 cells were subcutaneously implanted into 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice.
item258 For the subcutaneous implantation model, 1 × 107 cells were subcutaneously implanted into 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice.
item259 .
item260 .
item261 FtoKO mice were backcrossed to WT C57BL/6 mice to remove Cre and bred to homozygosity. Results are reported for male mice on the same genetic background (C57BL6/J). For the diet-induced bone loss studies, mice were fed a 60% high-fat diet (D12492, Research Diets) from 6 wk of age to 24 wk. Genotyping strategies are available upon request. NBD (KKKKKKKKGGTALDWSWLQTE) with the Trp to Ala substitutions designed to render the peptide inactive underlined, was a gift from D.C.G. and dissolved in water before use. Next, 10 mg/kg NBD was intraperitoneally injected in 29-wk old FtoOc KO mice every other day for 9 d. One day after the last injection, bone was harvested for analysis of DNA damage.
item262 3 × 106 treated HCC cells resuspended in 100 uL PBS were subcutaneously injected to the left flank of the mice, which were randomly divided into several groups. Tumor sizes were measured every 3 to 5 days.
item263 Mice were maintained at 22 ± 2 ℃ with a humidity of 35 ± 5% under a 12 h light and 12 h dark cycle, with free access to water and food. For the HFD experiment, female control (Ftoflox/flox) and adipose-selective fto knockout (Fabp4-Cre Ftoflox/flox, fto-AKO) mice were fed with high-fat diet (60% fat in calories; Research Diets, D12492) for the desired periods of time, and food intake and body weight were measured every week after weaning (at 3 weeks of age).
item264 Mice were maintained at 22 ± 2 ℃ with a humidity of 35 ± 5% under a 12 h light and 12 h dark cycle, with free access to water and food. For the HFD experiment, female control (Ftoflox/flox) and adipose-selective fto knockout (Fabp4-Cre Ftoflox/flox, fto-AKO) mice were fed with high-fat diet (60% fat in calories; Research Diets, D12492) for the desired periods of time, and food intake and body weight were measured every week after weaning (at 3 weeks of age).
item265 Mice were maintained at 22 ± 2 ℃ with a humidity of 35 ± 5% under a 12 h light and 12 h dark cycle, with free access to water and food. For the HFD experiment, female control (Ftoflox/flox) and adipose-selective fto knockout (Fabp4-Cre Ftoflox/flox, fto-AKO) mice were fed with high-fat diet (60% fat in calories; Research Diets, D12492) for the desired periods of time, and food intake and body weight were measured every week after weaning (at 3 weeks of age).
item266 Mice were maintained at 22 ± 2 ℃ with a humidity of 35 ± 5% under a 12 h light and 12 h dark cycle, with free access to water and food. For the HFD experiment, female control (Ftoflox/flox) and adipose-selective fto knockout (Fabp4-Cre Ftoflox/flox, fto-AKO) mice were fed with high-fat diet (60% fat in calories; Research Diets, D12492) for the desired periods of time, and food intake and body weight were measured every week after weaning (at 3 weeks of age).
item267 Mice were maintained at 22 ± 2 ℃ with a humidity of 35 ± 5% under a 12 h light and 12 h dark cycle, with free access to water and food. For the HFD experiment, female control (Ftoflox/flox) and adipose-selective fto knockout (Fabp4-Cre Ftoflox/flox, fto-AKO) mice were fed with high-fat diet (60% fat in calories; Research Diets, D12492) for the desired periods of time, and food intake and body weight were measured every week after weaning (at 3 weeks of age).
item268 Eight-to-twelve-week-old female C57BL/6 mice purchased from Jackson Laboratories were immunized subcutaneously at the base of the tail with 100 ug per mouse of OVA protein (Invivogen, vac-pova) adjuvanted with 25 ug of HMW vaccine grade Poly I:C (Invivogen, vac-pic), 25 ug of circular RNA alone or with in vivo-jetPEI (Polyplus Transfection, 201-10G), 25 ug of modified RNA alone or with in vivo-jet PEI.
item269 Eight-to-twelve-week-old female C57BL/6 mice purchased from Jackson Laboratories were immunized subcutaneously at the base of the tail with 100 ug per mouse of OVA protein (Invivogen, vac-pova) adjuvanted with 25 ug of HMW vaccine grade Poly I:C (Invivogen, vac-pic), 25 ug of circular RNA alone or with in vivo-jetPEI (Polyplus Transfection, 201-10G), 25 ug of modified RNA alone or with in vivo-jet PEI.
item270 The spleen in the upper left lateral abdomen of the anesthetized mice were exposed, 106 cells suspended in 20 uL phosphate-buffered saline (PBS) were injected into the distal tip of the spleen. After injection, replacing the spleen, and closing the incision.
item271 The spleen in the upper left lateral abdomen of the anesthetized mice were exposed, 106 cells suspended in 20 uL phosphate-buffered saline (PBS) were injected into the distal tip of the spleen. After injection, replacing the spleen, and closing the incision.
item272 200 uL PBS containing 1×107 cells of stable cells were subcutaneously injected into male BALB/c athymic nude mice (6-week old, 18-20 g).
item273 200 uL PBS containing 1×107 cells of stable cells were subcutaneously injected into male BALB/c athymic nude mice (6-week old, 18-20 g).
item274 Rats inhaled CB at dose of 0, 5 or 30 mg/m3 for 28 days, 6 h/day, respectively. The rats inhaled CB at dose of 0 or 30 mg/m3 for 14 days, 28 days and 90 days, respectively. In vitro experiments, the normal human bronchial epithelial cell line (16HBE) was treated with CB (0, 50, 100 and 200 ug/mL) for 24 h.
item275 .
item276 For the animal studies, 6-week-old male athymic BALB/c nude mice were implanted with modified ALKBH5 expressing Hct116 or RKO cells (105/ml, 200 ml) via lateral tail vein injection. All the mice were killed after 6 weeks, and the lungs were subjected to H&E staining.
item277 For subcutaneous tumor model, each mouse was injected subcutaneously in the right flank with 2 × 106 U87MG cells (METTL3-KD or control) in 100 uL PBS.
item278 For subcutaneous tumor model, each mouse was injected subcutaneously in the right flank with 2 × 106 U87MG cells (METTL3-KD or control) in 100 uL PBS.
item279 For subcutaneous tumor model, each mouse was injected subcutaneously in the right flank with 2 × 106 U87MG cells (METTL3-KD or control) in 100 uL PBS.
item280 For subcutaneous tumor model, each mouse was injected subcutaneously in the right flank with 2 × 106 U87MG cells (METTL3-KD or control) in 100 uL PBS.
item281 For subcutaneous tumor model, each mouse was injected subcutaneously in the right flank with 2 × 106 U87MG cells (METTL3-KD or control) in 100 uL PBS.
item282 For subcutaneous tumor model, each mouse was injected subcutaneously in the right flank with 2 × 106 U87MG cells (METTL3-KD or control) in 100 uL PBS.
item283 Induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days.
item284 Induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days.
item285 .
item286 Mice 8 weeks after splenic portal vein injection of BGC823 cells with METTL3 overexpression or vector-transfected cells.
item287 Mice 8 weeks after splenic portal vein injection of BGC823 cells with METTL3 overexpression or vector-transfected cells.
item288 .
item289 The luciferase signal intensity from days 7 to 42 is on equivalent scales in the models. Bioluminescent flux (photons/s/cm2/steradian) was determined for the lung metastases.
item290 .
item291 2 × 106 cells suspended in 40 uL PBS were injected into the inferior hemispleen into each 6-week-old BALB/c nude mouse.
item292 The transfected cells (2×106) were directly subcutaneously injected in to flank of mice. The width and length were measured every six days. After three weeks, the mice were killed and the necropsies were weighted.
item293 .
item294 .
item295 .
item296 .
item297 According to the method published previously , exponentially growing MG63 and MG63/DXR were harvested, counted with trypan blue to show cells with at least 95% viability, and then resuspended at a final amount from 5 × 106 to 5 × 103 in 100 uL Matrigel (Corning, NY, USA) before being injected subcutaneously into 5-week-old female nude mice.The mice were monitored once every 3 days until the 60th day, and euthanized humanely after the experiment
item298 According to the method published previously , exponentially growing MG63 and MG63/DXR were harvested, counted with trypan blue to show cells with at least 95% viability, and then resuspended at a final amount from 5 × 106 to 5 × 103 in 100 uL Matrigel (Corning, NY, USA) before being injected subcutaneously into 5-week-old female nude mice.The mice were monitored once every 3 days until the 60th day, and euthanized humanely after the experiment
item299 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item300 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item301 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item302 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item303 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item304 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item305 Mice were treated via nasal inhalation of adenovirus carrying Cre recombinase (5 × 106 p.f.u for Ad-Cre, Biowit Inc., Shenzhen, Guangdong), and were then killed at indicated times for gross inspection and histopathological examination.
item306 1 × 107 A549 cells were subcutaneously implanted into 4-week-old NOD/SCID mice.
item307 The experimental rats received intraperitoneal injection with IS at a dosage of 100 mg/kg/48 h for 8,16,24 weeks. The control rats (n = 5) received same volume of phosphate-buffered saline injection every 48 h for 8,16,24 weeks.
item308 .
item309 .
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item313 .
item314 Approximately 1 × 106 melanoma cells from each group were injected subcutaneously into the right side of the abdomen of BALB/c nude mice (male, 6 weeks old).
item315 .
item316 .
item317 For the engraftment experiments, 1×103 1×106 cells were injected into tail veins of non-irradiated 6-10 week-old female mice in 100 uL of DPBS per mouse. No blinding or randomization was applied to mice experiments. Routinely, each in vivo experiment was performed with three technical replicates (three mice per group) and independently repeated two to three times for each cell line.
item318 For the engraftment experiments, 1×103 1×106 cells were injected into tail veins of non-irradiated 6-10 week-old female mice in 100 uL of DPBS per mouse. No blinding or randomization was applied to mice experiments. Routinely, each in vivo experiment was performed with three technical replicates (three mice per group) and independently repeated two to three times for each cell line.
item319 For the engraftment experiments, 1×103 1×106 cells were injected into tail veins of non-irradiated 6-10 week-old female mice in 100 uL of DPBS per mouse. No blinding or randomization was applied to mice experiments. Routinely, each in vivo experiment was performed with three technical replicates (three mice per group) and independently repeated two to three times for each cell line.
item320 .
item321 For the orthotopic models, 2 × 106 cells with negative control (NC, sh-NC), sh-1 or sh-2 in 0.5 mL of PBS were subcutaneously injected into the dorsal flank of 2 mice respectively. Then 15 mice were separated into 3 groups (sh-NC, sh-1 and sh-2), of which the tumor pieces were tied to the base of the ceca. The growth of the tumors was monitored every 2 weeks after intraperitoneal injection of D-luciferin with a Xenogen IVIS 100 Bioluminescent Imaging System.
item322 DANCR KO or empty vector control were harvested and then mixed with matrigel (1:1) (BD Biosciences). Three different numbers of cells (1 × 104, 1 × 105, and 5 × 105 cells) were subcutaneously injected into nude mice, five animals per group.
item323 Equal number of PC-3 cells was injected subcutaneously into right flank.
item324 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item325 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item326 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item327 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item328 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item329 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item330 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item331 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item332 Mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into two groups (five mice per group) after the diameter of the xenografted tumors had reached approximately 5 mm in diameter. Xenografted mice were then administrated with PBS or DDP (3 mg/kg per day) for three times a week, and tumor volume were measured every second day.
item333 For subcutaneous transplanted model, control and miR-143-3p stable A549 cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 uL PBS + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient mice to investigate tumor growth.
item334 .
item335 .
item336 Six-week-old BALB/c nude mice were purchased from the College of Veterinary Medicine, Yang Zhou University. For the xenografted tumor model, 1 × 107 HCT116 cells in 0.2 mL PBS were subcutaneously injected into BALB/c nude mice, which were randomly divided into four groups (six mice per group). The volume of the tumors was calculated with the following equation: V = 0.5 × (length × width2). For metastasis experiments, 2 × 106 cells in 0.2 mL PBS were injected into the tail vein of nude mice, which were randomly divided into four groups (six mice per group).
item337 .
item338 5 × 106 cells were subcutaneously injected into the left or right flank of each mouse.
item339 .
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item347 .
item348 2 × 106 A549 cells stably transfected with shRNA-ALKBH5 were injected into the flank of male athymic BALB/c nude mice (4-5 weeks old, 10 mice).
item349 4-6 weeks old NOD/Shi-scid/IL-2Rgamma null (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG)) mice (female).
item350 4-6 weeks old NOD/Shi-scid/IL-2Rgamma null (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG)) mice (female).
item351 4-6 weeks old NOD/Shi-scid/IL-2Rgamma null (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG)) mice (female).
item352 .
item353 Cells transfected with miR-186 overexpression lentivirus (Lenti-miR-186), miR-186 inhibitor (Lenti-anti-miR-186), Lenti-miR-186 & METTL3 overexpression plasmid (Lenti-METTL3), Lenti-anti-miR-186 & METTL3 shRNA (sh-METTL3) or empty lentivirus control (Lenti-NC) were subcutaneously injected into the lower flank of nude mice.
item354 .
item355 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item356 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item357 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item358 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item359 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item360 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item361 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item362 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item363 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item364 Male SPF BALB/c mice (qls02-0202) were purchased from Qinglongshan animal breeding farm. Mice were sacrificed by CO2 asphyxiation and testes were obtained for following histopathological analyses.
item365 .
item366 .
item367 5 × 106 cells in PBS were injected subcutaneously into one side of the posterior flanks of Balb/C nude mice at 6-8 weeks old.
item368 Indicated stable 143B cells were subcutaneously injected into nude mice.
item369 Indicated stable 143B cells were subcutaneously injected into nude mice.
item370 .
item371 .
item372 .
item373 Stable down-regulated FTO cells were prepared in Eca-109 and KYSE150 and subcutaneously injected into the flank of nude mouse with 2 × 106 cells per mouse.
item374 LoVo cells (5 × 106 cells/ 200 uL PBS) stably transfected with METTL3 knockdown lentiviral vector or control vector were respectively injected subcutaneously into the left flank of each mouse.
item375 .
item376 .
item377 .
item378 .
item379 Gastric cancer cell line MGC803 (shRNA-NC or shKIAA1429; 1 × 107) was injected subcutaneously into the armpit of BALB/c nude mice (5-week-old, male, n = 4 for each group). Tumor growth was monitored at 3-day intervals.
item380 .
item381 .
item382 .
item383 For the experiments, mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into indicated groups (five mice per group). To assess the in vivo effects of cycloleucine, the xenografted tumors had reached approximately 5 mm in diameter from mice and then these xenografted mice were feed with Vehicle or cycloleucine (25 mg/kg twice weekly) and tumor volume were measured every 3 day. Tumor volume was estimated as 0.5 × a2 × b (where a and b represent a tumors short and long diameter, respectively). Mice were euthanized after 7 weeks and the tumors were measured a final time.
item384 For the experiments, mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into indicated groups (five mice per group). To assess the in vivo effects of cycloleucine, the xenografted tumors had reached approximately 5 mm in diameter from mice and then these xenografted mice were feed with Vehicle or cycloleucine (25 mg/kg twice weekly) and tumor volume were measured every 3 day. Tumor volume was estimated as 0.5 × a2 × b (where a and b represent a tumors short and long diameter, respectively). Mice were euthanized after 7 weeks and the tumors were measured a final time.
item385 For the experiments, mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into indicated groups (five mice per group). To assess the in vivo effects of cycloleucine, the xenografted tumors had reached approximately 5 mm in diameter from mice and then these xenografted mice were feed with Vehicle or cycloleucine (25 mg/kg twice weekly) and tumor volume were measured every 3 day. Tumor volume was estimated as 0.5 × a2 × b (where a and b represent a tumors short and long diameter, respectively). Mice were euthanized after 7 weeks and the tumors were measured a final time.
item386 .
item387 For liver metastasis model, mice were anaesthetized and an incision was made through the skin and peritoneum to expose the spleen. 1 × 106 HCT116 cells were injected into the spleen (n = 4 each group).
item388 For liver metastasis model, mice were anaesthetized and an incision was made through the skin and peritoneum to expose the spleen. 1 × 106 HCT116 cells were injected into the spleen (n = 4 each group).
item389 For the subcutaneous implantation model, UM-UC-3 cells (2 × 106 cells per mouse) stably METTL3 knocked down (shMETTL3-1, shMETTL3-2) were injected into the flanks of mice.
item390 For the subcutaneous implantation model, UM-UC-3 cells (2 × 106 cells per mouse) stably METTL3 knocked down (shMETTL3-1, shMETTL3-2) were injected into the flanks of mice.
item391 For the subcutaneous implantation model, UM-UC-3 cells (2 × 106 cells per mouse) stably METTL3 knocked down (shMETTL3-1, shMETTL3-2) were injected into the flanks of mice.
item392 For the subcutaneous implantation model, UM-UC-3 cells (2 × 106 cells per mouse) stably METTL3 knocked down (shMETTL3-1, shMETTL3-2) were injected into the flanks of mice.
item393 The stable transfection of SCC25 cells (1 × 107 cells in 0.1 mL) with lenti-sh-METTL3 or blank vectors was injected subcutaneously into BALB/c nude mice.
item394 The stable transfection of SCC25 cells (1 × 107 cells in 0.1 mL) with lenti-sh-METTL3 or blank vectors was injected subcutaneously into BALB/c nude mice.
item395 All mice were housed at 21℃ ± 1℃ with a humidity of 55% ± 10% and a 12-hour light/dark cycle. The high-fat diets (HFDs), containing 60% kcal from fat, 20% kcal from carbohydrate, and 20% kcal from protein.
item396 All mice were housed at 21℃ ± 1℃ with a humidity of 55% ± 10% and a 12-hour light/dark cycle. The high-fat diets (HFDs), containing 60% kcal from fat, 20% kcal from carbohydrate, and 20% kcal from protein.
item397 All mice were housed at 21℃ ± 1℃ with a humidity of 55% ± 10% and a 12-hour light/dark cycle. The high-fat diets (HFDs), containing 60% kcal from fat, 20% kcal from carbohydrate, and 20% kcal from protein.
item398 All mice were housed at 21℃ ± 1℃ with a humidity of 55% ± 10% and a 12-hour light/dark cycle. The high-fat diets (HFDs), containing 60% kcal from fat, 20% kcal from carbohydrate, and 20% kcal from protein.
item399 Male BALB/c nude mice (5 weeks old) were subcutaneously inoculated in the right axillary fossa with 200 uL (1 × 106 cells) of SIRT1-overexpressing Hep3B cells, SIRT1- and FTO-overexpressing Hep3B cells, shRNA-SIRT1-transfected HepG2 cells, shRNA-SIRT1- and shRNA-FTO-transfected HepG2 cells, and control cells.
item400 .
item401 .
item402 1×106 ES-2-Scrambled or ES-2-shRHPN1-AS1 cells were injected subcutaneously on the right side of the mice.
item403 .
item404 ,
item405 ,
item406 Injected YY1BM intratumorally into ESCC tumors grafted in nude mice and analyzed the survival time.
item407 A total of 2 × 106 GC cells were injected into the flank of nude mice in a 1:1 suspension of BD Matrigel (BD Biosciences) in phosphate-buffered saline (PBS) solution.
item408 A total of 2 × 106 GC cells were injected into the flank of nude mice in a 1:1 suspension of BD Matrigel (BD Biosciences) in phosphate-buffered saline (PBS) solution.
item409 A total of 2 × 106 GC cells were injected into the flank of nude mice in a 1:1 suspension of BD Matrigel (BD Biosciences) in phosphate-buffered saline (PBS) solution.
item410 .
item411 .
item412 .
item413 For the unilateral ureteral obstruction (UUO) model, male C57BL/6J mice at 8 weeks of age (20-22 g body weight) were first anaesthetized with pentobarbital sodium (50 mg/kg) via intraperitoneal injection. Then, the left ureter was ligated using 3-0 silk and a left lateral incision.
item414 Both sh-control and sh- ERRγ HepG2/ADR cells (5 × 106 per mouse, n=5 for each group) were diluted in 200uL PBS + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient mice.
item415 Both sh-control and sh- ERRγ HepG2/ADR cells (5 × 106 per mouse, n=5 for each group) were diluted in 200uL PBS + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient mice.
item416 Both sh-control and sh- ERRγ HepG2/ADR cells (5 × 106 per mouse, n=5 for each group) were diluted in 200uL PBS + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient mice.
item417 .
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item427 1 × 106 HCT-116-Luc-LINC00266-1-ORF-Flag, HCT-116-Luc- LINC00266-1-5'U, or HCT-116-Luc-LINC00266-1-MT cells were subcutaneously injected into the dorsal flanks of each BALB/c nude mouse (n = 6).
item428 .
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item430 All the mice were randomly divided into five groups of four mice each, including PC3-shNC, PC3-shMETTL3-1/2, LNCaP-vector and LNCaP-METTL3 group.
item431 .
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item434 A longitudinal incision was made on the skin and the muscles were separated to expose the femur. A transverse osteotomy was performed in the mid-diaphysis of the femur, and the bones were stabilized by inserting a 23-gauge intramedullary needle. Equal amounts (100 uL) of phosphate-buffered saline (PBS), plasmid METTL3 and agomiR-7212-5p (10 mg/kg body weight) were locally injected into the femoral fracture site.
item435 Established cohorts of mice bearing tumour xenografts driven by PTEN-deficient BxPC-3 and PANC-1 cells with PIK3CB overexpression. When tumours grew to ~300 mm3, mice were grouped and administered with vehicle (DMSO) or KIN-193 via intraperitoneal injection (20 mg/kg) once daily.
item436 Established cohorts of mice bearing tumour xenografts driven by PTEN-deficient BxPC-3 and PANC-1 cells with PIK3CB overexpression. When tumours grew to ~300 mm3, mice were grouped and administered with vehicle (DMSO) or KIN-193 via intraperitoneal injection (20 mg/kg) once daily.
item437 Established cohorts of mice bearing tumour xenografts driven by PTEN-deficient BxPC-3 and PANC-1 cells with PIK3CB overexpression. When tumours grew to ~300 mm3, mice were grouped and administered with vehicle (DMSO) or KIN-193 via intraperitoneal injection (20 mg/kg) once daily.
item438 Established cohorts of mice bearing tumour xenografts driven by PTEN-deficient BxPC-3 and PANC-1 cells with PIK3CB overexpression. When tumours grew to ~300 mm3, mice were grouped and administered with vehicle (DMSO) or KIN-193 via intraperitoneal injection (20 mg/kg) once daily.
item439 .
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item441 Female athymic BALB/c nude mice (4-week-old) were provided (SLAC Laboratory Animal Co. Ltd.). The animals were raised in a pathogen-free animal laboratory and randomly divided into the control or experimental group (six mice in each group).
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item446 For the drug-resistant subcutaneous tumor models, drug administration was adopted when the tumors reached about 50 mm3 in size, at which point mice were randomized for treatment with DMSO(intraperitoneally) or sorafenib (50 mg/kg/every 2 days, intraperitoneally). For the patient-derived tumor xenograft model, drug administration began 4 weeks after tumors reached about 100 mm3 in size with sorafenib (50 mg/kg/every 3 days, intraperitoneally) or siCtrl/siMETTL3 intratumor injection.
item447 .
item448 A total of 2 × 106 cells was mixed with 0.2 ml PBS (pH 7.4) and 30% (v/v) Matrigel matrix (BD Biosciences).
item449 A total of 2 × 106 cells was mixed with 0.2 ml PBS (pH 7.4) and 30% (v/v) Matrigel matrix (BD Biosciences).
item450 .
item451 gRNA and Cas9 mRNA were mixed at concentration of 50 and 100 ng/ul, respectively, and injected to the cytoplasm of one-cell-stage embryos of C57BL/6 genetic background.
item452 Congenic recipient mice (CD45.2) at 8-10 weeks old were used for AML transplantation, and CD45.1 recipients at 8-10 weeks old were used for normal hematopoietic transplantation assays.
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item460 About 5 × 106 cells transfected with pc-YTHDF1 in SHG-44 cell (si-YTHDF1 in U87 cell) were suspended in 0.1 mL of PBS and injected subcutaneously into nude mice.
item461 The OE21 cells were stably transfected with sh-ALKBH5 (#1), sh-ALKBH5 (#2) or sh-scramble as negative control and injected (2 × 106 cells/mouse in 100 uL volume) subcutaneously into the back of male athymic BALB/c nude mice (6 weeks old, Japan SLC).
item462 1×106/mouse cells were suspended in 50 uL of serum-free DMEM and subcutaneously injected into the flank of each mouse. Tumor diameters and volume (length × width2 × 0.5236) were calculated every other day using calipers.
item463 METTL3 stable knockdown or overexpression HCT116 cells were collected and resuspended at a density of 5 × 106 or 3 × 106 cells per 150 uL PBS.
item464 .
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item468 2 × 106 transfected HCT116 cells in 0.2 ml PBS were injected into the tail vein of nude mice which were randomly divided into nine groups (eight mice per group).
item469 The nude mice were randomly grouped into 2 groups, of 5 mice each; 786-0 cells (7×106 in 100 L PBS) were stabilized with ALKBH5 knockdown lentiviral transfection vector (shALKBH5) or scramble vector (SCR) via subcutaneous injection into the left armpit of each mouse.
item470 .
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item472 After a median incision on the neck, the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were isolated. The left CCA and the ECA were ligated.
item473 Female nude, athymic, BALB/c-nu/nu mice (6-7 weeks old; Harlan) were injected subcutaneously (s.c.) with serially diluted numbers (5,000, 10,000, or 20,000) of OVCAR5_GFP or OVCAR5_FTO cells mixed with Matrigel (Fisher Scientific). Mice were monitored bi-weekly and time to tumor initiation and tumor growth were recorded.
item474 A total of 5.0 × 106 normal (shNC) or METTL3-depleted (shMETTL3) CAL-27 cells were inoculated subcutaneously into the dorsal flank of the nude mice in 100 uL PBS.
item475 A total of 5.0 × 106 normal (shNC) or METTL3-depleted (shMETTL3) CAL-27 cells were inoculated subcutaneously into the dorsal flank of the nude mice in 100 uL PBS.
item476 .
item477 1 × 107 Bel-7404 cells in 200 uL PBS were injected into the right flank of nude mice.
item478 .
item479 .
item480 .
item481 Previously prepared U87 cells (5 × 106 cells, 60 uL) stably infected with miRNA were injected subcutaneously in the right flank of the mice, whereas control U87 cells infected with empty vector were injected in the left flank.
item482 Previously prepared U87 cells (5 × 106 cells, 60 uL) stably infected with miRNA were injected subcutaneously in the right flank of the mice, whereas control U87 cells infected with empty vector were injected in the left flank.
item483 .
item484 The NOD-SCID mice were intravenously (tail vein) implanted with sh-RNA-established NB4 cells. Direct injection of 5 × 106 shRNA-transformed NB4 cells into 150 uL of PBS was performed to establish intravenous (tail vein) leukemia.
item485 The NOD-SCID mice were intravenously (tail vein) implanted with sh-RNA-established NB4 cells. Direct injection of 5 × 106 shRNA-transformed NB4 cells into 150 uL of PBS was performed to establish intravenous (tail vein) leukemia.
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item493 2 × 106 cells were suspended in 200 uL of serum-free RPMI 1640 for injection into each mouse.
item494 2 × 106 cells were suspended in 200 uL of serum-free RPMI 1640 for injection into each mouse.
item495 2 × 106 cells were suspended in 200 uL of serum-free RPMI 1640 for injection into each mouse.
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item511 Male mice were subcutaneously injected with tumour cells near the limbs to establish xenografts (1 × 106/mouse, 0.2 mL for each injection site; METTL3-overexpressing TCam-2/CDDP cells were inoculated once at the initial time and IGF2BP1-inhibited TCam-2/CDDP cells were inoculated every 3 days).
item512 Male mice were subcutaneously injected with tumour cells near the limbs to establish xenografts (1 × 106/mouse, 0.2 mL for each injection site; METTL3-overexpressing TCam-2/CDDP cells were inoculated once at the initial time and IGF2BP1-inhibited TCam-2/CDDP cells were inoculated every 3 days).
item513 .
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item517 A method of randomisation was used to determine the experimental groups. In total, 78 female BALB/c nu/nu mice (4-6-week-old) were selected at random and were divided into different groups. A total of 5 × 106 ISK cells or 1 × 104 ECSCisk were suspended in 100 uL of PBS and then were injected into the mice. After 2 weeks, the presence of tumours was examined. ISK cells (5 × 106, 5 × 105, 5 × 104, 1 × 104, or 1 × 103) and ECSCisk (1 × 104, 1 × 103, 1 × 102, 10, or 1) were injected and analysed for their abilities to form xenograft tumours. After 4 weeks, subsequent experiments were performed.
item518 To establish xenograft model, RKO cells (1 × 107) tansduced with si-control (si-NC) or si-ALKBH5 or si-ALKBH5+NEAT1 were injected subcutaneously into the right flank of the nude mice (n = 6 each group) every 5 days for 4 times.
item519 .
item520 .
item521 Rats were anesthetized and incised through the midline of the abdomen, and the left renal vertebral arch and arteries were blocked for 45 min, thereby resulting in left kidney ischemia. At the same time, the right kidney was removed, further aggravating the degree of left kidney injury.
item522 Rats were anesthetized and incised through the midline of the abdomen, and the left renal vertebral arch and arteries were blocked for 45 min, thereby resulting in left kidney ischemia. At the same time, the right kidney was removed, further aggravating the degree of left kidney injury.
item523 Rats were anesthetized and incised through the midline of the abdomen, and the left renal vertebral arch and arteries were blocked for 45 min, thereby resulting in left kidney ischemia. At the same time, the right kidney was removed, further aggravating the degree of left kidney injury.
item524 .
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item527 .
item528 .
item529 The specified number of viable SKOV3/DDP cells and SKOV3/DDP cells with TRIM29 knock down were resuspended in 100 uL PBS, injected subcutaneously under the left and right back of 4-week old nude mice respectively (n = 3 per group).
item530 A total of 1 × 107 control or KCNMB2-AS1-depleted SiHa cells were resuspended in 0.1 ml phosphate-buffered saline and inoculated into the armpit of 5-week-old male BALB/c nude mice.
item531 The GC cell line MKN-45 stably infected with lentivirus expressing sh-HBXIP was prepared into 5 × 107 cells/mL cell suspension.
item532 .
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item535 .
item536 Under anesthesia with ether, the nude mice were disinfected and subcutaneously inoculated with cells transfected with oe-NC, oe-KDM5A + oe-NC and oe-KDM5A + oe-MOB3B at a density of 1 × 106 cells/mouse (200 uL) at the back of the right hind leg.
item537 .
item538 .
item539 Behavioral tests and staining were performed to evaluate the damage to the diabetic hippocampus in model rats.
item540 .
item541 Inducing hepatic fibrosis in C57/BL6 mice by intraperitoneal injection of CCl4. The reversal model of hepatic fibrosis was established by stopping drug after continuous injection of CCl4. Dynamic m6A methylation was evaluated using MeRIP-Seq in the progression and reversal of hepatic fibrosis at different stages.
item542 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item543 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item544 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item545 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item546 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item547 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item548 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item549 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item550 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item551 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item552 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item553 NSCLC gefitinib-resistant cells (5 × 106 cells in 100 uL PBS) were injected subcutaneously into the lateral surface of the left abdomen of 6-week-old female BALB/c nude mice (at least five mice per group to ensure accuracy).
item554 RC cells SW480 at logarithmic growth phase were prepared into cell suspension with a concentration of about 1 × 107/100 L, which was then injected into the left axilla of nude mice with a 1 ml syringe to establish a subcutaneous mouse xenograft model. Once the tumor volume reached about 50 mm3, the nude mice were injected with miR-96 antagomir or NC antagomir (10 nmol once every 5 days for 5 weeks). After 5 weeks, the mice were euthanized, after which the subcutaneous transplanted tumor was removed, and weighed.
item555 RC cells SW480 at logarithmic growth phase were prepared into cell suspension with a concentration of about 1 × 107/100 L, which was then injected into the left axilla of nude mice with a 1 ml syringe to establish a subcutaneous mouse xenograft model. Once the tumor volume reached about 50 mm3, the nude mice were injected with miR-96 antagomir or NC antagomir (10 nmol once every 5 days for 5 weeks). After 5 weeks, the mice were euthanized, after which the subcutaneous transplanted tumor was removed, and weighed.
item556 In vivo myocardial I/R injury was performed by 60 min of ligation of LAD followed by 5 h of reperfusion.
item557 In vivo myocardial I/R injury was performed by 60 min of ligation of LAD followed by 5 h of reperfusion.
item558 .
item559 The first CDX generation was constructed in 4-6 week-old male BALB/c nude mice and treated with sorafenib (30 mg/kg daily, oral gavage). Twelve weeks later, the most resistant xenograft was disaggregated and implanted subcutaneously into 4-6 week-old BALB/c nude mice as the second SR-CDX. Four weeks after implantation, the second SR-CDX mice were treated with sorafenib (30 mg/kg daily, oral gavage) and locally injected with sh-circRNA-SORE lentivirus or its negative control (twice a week for 2 weeks).
item560 Two hundred milliliters of A549 cells (1 × 106) were injected into the left flank of the back of each mouse.
item561 Two hundred milliliters of A549 cells (1 × 106) were injected into the left flank of the back of each mouse.
item562 Nude mice were subjected to a subcutaneous injection of 5× 106 control and ZFAS1 silencing CaSki cells suspended in 0.2 mL DMEM medium.
item563 Thirty-two 6 week-old female nude mice divided into four groups (n = 8 per group) for injections of H1299 cells transfected with (a) pLKO.1 + pWPI, (b) shcircNOTCH1 + pWPI, (c) pLKO.1 + oeGPER and (d) shcircNOTCH1 + oeGPER.
item564 .
item565 Stable DMDRMR knockdown (KD) and control cell lines were injected subcutaneously (s.c.; 1 × 107 cells/inoculum) into the flanks of recipient NOD/SCID/IL2Rγ-null (NSG) mice.
item566 Stable DMDRMR knockdown (KD) and control cell lines were injected subcutaneously (s.c.; 1 × 107 cells/inoculum) into the flanks of recipient NOD/SCID/IL2Rγ-null (NSG) mice.
item567 .
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item570 This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG).
item571 This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG).
item572 This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG).
item573 This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG).
item574 This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG).
item575 This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG).
item576 .
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item585 When xenografted tumor growth reached 500 mm3, the mice were subjected to intratumoral injection of Ad-con or Ad-HNF3γ every other day. For the PDX model, fresh patient HCC tissues were cut into fragments with a volume of 3 × 3 mm3 and then implanted subcutaneously into the flanks of nude mice. The mice were given sorafenib (30 mg/kg) or vehicle orally twice a week for 24 days.
item586 .
item587 .
item588 Mice were randomly divided into three groups, and subcutaneously injected with control, circ-ARL3-silenced and circ-ARL3&miR-1305-silenced HepG2.2.15 cells.
item589 Mice were randomly divided into three groups, and subcutaneously injected with control, circ-ARL3-silenced and circ-ARL3&miR-1305-silenced HepG2.2.15 cells.
item590 Mice were randomly divided into three groups, and subcutaneously injected with control, circ-ARL3-silenced and circ-ARL3&miR-1305-silenced HepG2.2.15 cells.
item591 .
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item597 Cell suspensions (2 × 106 cells/mL) made with MCF-7/ADR cells stably expressing METTL3 and/or miR-221-3p inhibitor were subcutaneously implanted into each mouse. One week later, xenografted mice were injected with 0.1 mL ADR (25 mg/kg, intraperitoneal injection) twice a week.
item598 Cell suspensions (2 × 106 cells/mL) made with MCF-7/ADR cells stably expressing METTL3 and/or miR-221-3p inhibitor were subcutaneously implanted into each mouse. One week later, xenografted mice were injected with 0.1 mL ADR (25 mg/kg, intraperitoneal injection) twice a week.
item599 Cell suspensions (2 × 106 cells/mL) made with MCF-7/ADR cells stably expressing METTL3 and/or miR-221-3p inhibitor were subcutaneously implanted into each mouse. One week later, xenografted mice were injected with 0.1 mL ADR (25 mg/kg, intraperitoneal injection) twice a week.
item600 Cell suspensions (2 × 106 cells/mL) made with MCF-7/ADR cells stably expressing METTL3 and/or miR-221-3p inhibitor were subcutaneously implanted into each mouse. One week later, xenografted mice were injected with 0.1 mL ADR (25 mg/kg, intraperitoneal injection) twice a week.
item601 Cell suspensions (2 × 106 cells/mL) made with MCF-7/ADR cells stably expressing METTL3 and/or miR-221-3p inhibitor were subcutaneously implanted into each mouse. One week later, xenografted mice were injected with 0.1 mL ADR (25 mg/kg, intraperitoneal injection) twice a week.
item602 Athymic BALB/c mice were injected with LCSC cells at the armpit area subcutaneously. The mice were then sacrificed and the tumors recovered.
item603 The in vivo experiment method for transplantation of tumors was subcutaneous injection of 1 × 107 ALKBH5-stable knockdown C918 cells into BALB/c nude mice.
item604 Three-week-old BABL/c female nude mice were randomized into three groups. 5 × 106 143B cells were subcutaneously injected in mice, and the tumor volume was assessed every 2 weeks. Eight weeks after injection, the animals were killed. The xenograft tumors were harvested and the tumor volumes were calculated by the standard formula: length × width2/2.
item605 Three-week-old BABL/c female nude mice were randomized into three groups. 5 × 106 143B cells were subcutaneously injected in mice, and the tumor volume was assessed every 2 weeks. Eight weeks after injection, the animals were killed. The xenograft tumors were harvested and the tumor volumes were calculated by the standard formula: length × width2/2.
item606 Three-week-old BABL/c female nude mice were randomized into three groups. 5 × 106 143B cells were subcutaneously injected in mice, and the tumor volume was assessed every 2 weeks. Eight weeks after injection, the animals were killed. The xenograft tumors were harvested and the tumor volumes were calculated by the standard formula: length × width2/2.
item607 Three-week-old BABL/c female nude mice were randomized into three groups. 5 × 106 143B cells were subcutaneously injected in mice, and the tumor volume was assessed every 2 weeks. Eight weeks after injection, the animals were killed. The xenograft tumors were harvested and the tumor volumes were calculated by the standard formula: length × width2/2.
item608 Three-week-old BABL/c female nude mice were randomized into three groups. 5 × 106 143B cells were subcutaneously injected in mice, and the tumor volume was assessed every 2 weeks. Eight weeks after injection, the animals were killed. The xenograft tumors were harvested and the tumor volumes were calculated by the standard formula: length × width2/2.
item609 A549 cells were transfected with the pZW1-FCS-circNDUFB2 plasmid or pZW1-FCS-Vector plasmid, and selected with G418 (800 ug/ml) for 4 weeks, and then 2 × 106 A549 cells were subcutaneously injected into the right flank of each mouse.
item610 A549 cells were transfected with the pZW1-FCS-circNDUFB2 plasmid or pZW1-FCS-Vector plasmid, and selected with G418 (800 ug/ml) for 4 weeks, and then 2 × 106 A549 cells were subcutaneously injected into the right flank of each mouse.
item611 A549 cells were transfected with the pZW1-FCS-circNDUFB2 plasmid or pZW1-FCS-Vector plasmid, and selected with G418 (800 ug/ml) for 4 weeks, and then 2 × 106 A549 cells were subcutaneously injected into the right flank of each mouse.
item612 .
item613 Exosc10cKO_Emx1-Cre mouse embryos were subjected to the p53-inhibitor Pifithrin-Alpha (PFT-Alpha) by intraperitoneal injection of 2.2 mg/kg PFT-Alpha (Selleckchem) into the pregnant mother at between E9.5 and E12.5 or between E9.5 and E15.5. Embryonic brains were isolated at E13.5 or E18.5 and immunohistochemistry was performed.
item614 The enriched mammosphere cells derived from engineered BT549 and Hs578T with silenced lncRNA KB-1980E6.3 (shKB/vector), BT549, and Hs578T with lncRNA KB-1980E6.3 knockdown combined with ectopic c-Myc (shKB/c-Myc), BT549, and Hs578T with silenced IGF2BP1 (shIGF2BP1/vector), BT549, and Hs578T with knocked down IGF2BP1 combined with ectopic c-Myc (shIGF2BP1/c-Myc), and BT549, and Hs578T/shNC/vector control cells were used in Xenograft experiments. Three doses (1 × 105, 1 × 104 and 1 × 103) of spheres derived from the engineered Hs578T and 1 × 105 of spheres derived from the engineered BT549 were subcutaneously inoculated into 4- to 6-week-old female nude mice (n = 5 per group). Mice were then treated with either bevacizumab (10 mg/kg every 3 days) to form a hypoxic tumor microenvironment or vehicle PBS to form a non-hypoxic condition
item615 The enriched mammosphere cells derived from engineered BT549 and Hs578T with silenced lncRNA KB-1980E6.3 (shKB/vector), BT549, and Hs578T with lncRNA KB-1980E6.3 knockdown combined with ectopic c-Myc (shKB/c-Myc), BT549, and Hs578T with silenced IGF2BP1 (shIGF2BP1/vector), BT549, and Hs578T with knocked down IGF2BP1 combined with ectopic c-Myc (shIGF2BP1/c-Myc), and BT549, and Hs578T/shNC/vector control cells were used in Xenograft experiments. Three doses (1 × 105, 1 × 104 and 1 × 103) of spheres derived from the engineered Hs578T and 1 × 105 of spheres derived from the engineered BT549 were subcutaneously inoculated into 4- to 6-week-old female nude mice (n = 5 per group). Mice were then treated with either bevacizumab (10 mg/kg every 3 days) to form a hypoxic tumor microenvironment or vehicle PBS to form a non-hypoxic condition
item616 .
item617 A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks (n = 6/group).
item618 A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks (n = 6/group).
item619 A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks (n = 6/group).
item620 A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks (n = 6/group).
item621 A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks (n = 6/group).
item622 .
item623 .
item624 Groups of HCT116-Luc-shCtrl, HCT116-Luc-shLINC00460, and HCT116-Luc-shLINC00460 + HMGA1 cells (5 × 106) were injected subcutaneously into the flanks of mice correspondingly.
item625 Groups of HCT116-Luc-shCtrl, HCT116-Luc-shLINC00460, and HCT116-Luc-shLINC00460 + HMGA1 cells (5 × 106) were injected subcutaneously into the flanks of mice correspondingly.
item626 Groups of HCT116-Luc-shCtrl, HCT116-Luc-shLINC00460, and HCT116-Luc-shLINC00460 + HMGA1 cells (5 × 106) were injected subcutaneously into the flanks of mice correspondingly.
item627 MCF-7 cells transfected with sh-LINC00958 or empty vector were resuspended at 2 × 107 cells/mL.
item628 The mice were housed under standardized conditions and received normal diet. The mice were randomly divided into 5 groups (n = 6/each group): (1) Control group: normal mice served as control; (2) TAC-HF group: mice were subjected to transverse aortic constriction (TAC) to establish HF mouse model; (3) Sham group: sham-operated mice were subjected the same surgery without the placement of a ligature; (4) Doxorubicin-HF group: mice were intraperitoneally injected with doxorubicin to construct HF mouse model; and (5) PBS: mice were intraperitoneally injected with equal PBS as control.
item629 .
item630 .
item631 .
item632 .
item633 .
item634 Used 1 × 106 SNHG3 knocked down KY-SE150 cells and NC lentivirus to inject into the right flank of mice to generate xenografts.
item635 Used 1 × 106 SNHG3 knocked down KY-SE150 cells and NC lentivirus to inject into the right flank of mice to generate xenografts.
item636 HCC cells (1 × 106/100 uL PBS) were administered to 4-week-old female BALB/c nude mice by subcutaneous injection (n = 6).
item637 HCC cells (1 × 106/100 uL PBS) were administered to 4-week-old female BALB/c nude mice by subcutaneous injection (n = 6).
item638 For tumor growth studies, whether in vivo RBM15 knockdown/overexpression experiments or in vivo rescue experiments, each group included six mice. Each mouse was injected with 100 uL of lentivirus-transfected tumor cells.
item639 For tumor growth studies, whether in vivo RBM15 knockdown/overexpression experiments or in vivo rescue experiments, each group included six mice. Each mouse was injected with 100 uL of lentivirus-transfected tumor cells.
item640 For tumor growth studies, whether in vivo RBM15 knockdown/overexpression experiments or in vivo rescue experiments, each group included six mice. Each mouse was injected with 100 uL of lentivirus-transfected tumor cells.
item641 For tumor growth studies, whether in vivo RBM15 knockdown/overexpression experiments or in vivo rescue experiments, each group included six mice. Each mouse was injected with 100 uL of lentivirus-transfected tumor cells.
item642 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
item643 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
item644 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
item645 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
item646 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
item647 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
item648 The SKOV3 ovarian cancer cell line was transfected with LV2-1 or LV2-NC. Thereafter, BALB/c nude mice (6-week old) were intraperitoneally injected with SKOV3 cells. The mice were killed after 5 weeks, and the number of ascites was determined.
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item661 The rats were adaptively fed for 1 week and then subcutaneous injection of complete Freund's adjuvant into the left hindfoot toe to establish an adjuvant arthritis (AA) rat model. After induction of the immune response for 20 days, all rats were anesthetized by the intraperitoneal injection of 1% sodium pentobarbital (60 mg/kg) and sacrificed by exsanguination of the abdominal aorta.
item662 The rats were adaptively fed for 1 week and then subcutaneous injection of complete Freund's adjuvant into the left hindfoot toe to establish an adjuvant arthritis (AA) rat model. After induction of the immune response for 20 days, all rats were anesthetized by the intraperitoneal injection of 1% sodium pentobarbital (60 mg/kg) and sacrificed by exsanguination of the abdominal aorta.
item663 The rats were adaptively fed for 1 week and then subcutaneous injection of complete Freund's adjuvant into the left hindfoot toe to establish an adjuvant arthritis (AA) rat model. After induction of the immune response for 20 days, all rats were anesthetized by the intraperitoneal injection of 1% sodium pentobarbital (60 mg/kg) and sacrificed by exsanguination of the abdominal aorta.
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item668 The mice were randomly divided into groups before subcutaneous injection with cells (107) suspended in 200 uL of phosphate-buffered saline. Tumors were measured on day 7 and then once every 7 days until 28 days after injection.
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item691 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of six-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck millipore, 217 503), then the C2C12 cells, which stably overexpressed METTL3 or GFP control, were injected to the injury site of mice on the following day. The regenerated muscles were collected at day 7 post-injection, frozen in liquid nitrogen for RNA and protein extraction.
item692 10 rats were divided into control and HPH group. In detail, 5 rats of the hypoxia groups were exposed to hypoxia (10%O2) chamber (AiPu XBS-02B, China) for 4 weeks. In addition, 5 rats of control group were kept under normoxic conditions (21% O2) for 4 weeks. Rats were housed in standard polypropylene cages under controlled photocycle (12 h light/12 h dark) under 22-25 ℃ temperature.
item693 10 rats were divided into control and HPH group. In detail, 5 rats of the hypoxia groups were exposed to hypoxia (10%O2) chamber (AiPu XBS-02B, China) for 4 weeks. In addition, 5 rats of control group were kept under normoxic conditions (21% O2) for 4 weeks. Rats were housed in standard polypropylene cages under controlled photocycle (12 h light/12 h dark) under 22-25 ℃ temperature.
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item697 In the control mice or ADR mice group, the parental or chemo-resistant K562 cells were infected with LV-shCtrl. In the ADR + shLINC00470 group, the chemo-resistant K562 cells were infected with LV- shLINC00470. These cells were injected, respectively, into these 5-week-old mice subcutaneously.
item698 In the control mice or ADR mice group, the parental or chemo-resistant K562 cells were infected with LV-shCtrl. In the ADR + shLINC00470 group, the chemo-resistant K562 cells were infected with LV- shLINC00470. These cells were injected, respectively, into these 5-week-old mice subcutaneously.
item699 BALB/c nude mice (4 weeks old) were acquired from Vital River Laboratory (Beijing, China). HCT116 cells with stable circ1662 expression (2 × 106 in 100 L of PBS) were injected via the tail vein. After 45 days, the mice were sacrificed. The lung metastatic carcinoma specimens were processed into paraffin-embedded sections for subsequent H&E staining and IHC.
item700 BALB/c nude mice (4 weeks old) were acquired from Vital River Laboratory (Beijing, China). HCT116 cells with stable circ1662 expression (2 × 106 in 100 L of PBS) were injected via the tail vein. After 45 days, the mice were sacrificed. The lung metastatic carcinoma specimens were processed into paraffin-embedded sections for subsequent H&E staining and IHC.
item701 BALB/c nude mice (4 weeks old) were acquired from Vital River Laboratory (Beijing, China). HCT116 cells with stable circ1662 expression (2 × 106 in 100 L of PBS) were injected via the tail vein. After 45 days, the mice were sacrificed. The lung metastatic carcinoma specimens were processed into paraffin-embedded sections for subsequent H&E staining and IHC.
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item711 BGC-823 cells (5 × 106) with shRNAs targeting YTHDF1 (sh-YTHDF1) or shRNAs targeting control (sh-NC) were trypsinized and suspended in 0.1 mL PBS and injected subcutaneously into the BALB/c mice (n = 5 mice per group).
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item714 Balb/c female nude mice at 4-6 weeks old were injected with 4 × 106 cells subcutaneously on the back.
item715 HCC cells (5 × 106 cells/mouse) that had been transfected with oe-NC + sh-NC, oe-KDM5B + sh-NC, or oe-KDM5B + sh-ITGA6, or treated with NS, GSK-467 (a selective inhibitor of KDM5B) + oe-NC or GSK-467 + oe-ITGA6 were then subcutaneously implanted into the back of mice.
item716 .
item717 As cells (5 million) in Matrigel or As-T (1 million) cells in PBS with or without gene manipulations were injected subcutaneously into the right flanks of female mice (6-8 weeks of age). For treatment with CS1 or CS2, As-T cells (1 million) in PBS were injected subcutaneously into the right flanks of 6-week-old female nude mice.
item718 As cells (5 million) in Matrigel or As-T (1 million) cells in PBS with or without gene manipulations were injected subcutaneously into the right flanks of female mice (6-8 weeks of age). For treatment with CS1 or CS2, As-T cells (1 million) in PBS were injected subcutaneously into the right flanks of 6-week-old female nude mice.
item719 As cells (5 million) in Matrigel or As-T (1 million) cells in PBS with or without gene manipulations were injected subcutaneously into the right flanks of female mice (6-8 weeks of age). For treatment with CS1 or CS2, As-T cells (1 million) in PBS were injected subcutaneously into the right flanks of 6-week-old female nude mice.
item720 As cells (5 million) in Matrigel or As-T (1 million) cells in PBS with or without gene manipulations were injected subcutaneously into the right flanks of female mice (6-8 weeks of age). For treatment with CS1 or CS2, As-T cells (1 million) in PBS were injected subcutaneously into the right flanks of 6-week-old female nude mice.
item721 DLD-1 cells were subcutaneously implanted into 4-6 weeks old female nude mice. When tumors reached a size of about 50 mm3, the nude mice were randomly divided into 6 groups.
item722 DLD-1 cells were subcutaneously implanted into 4-6 weeks old female nude mice. When tumors reached a size of about 50 mm3, the nude mice were randomly divided into 6 groups.
item723 Twelve female BALB/c nude mice (aged 4 weeks, 18-22g) were randomly divided into 2 groups. Stable circMETTL3-expression SUM1315 cells or control cells (1×106 cells in 0.1 mL PBS) was subcutaneously injected into mammary fat pads of the mice and the growth of tumors was followed up every week. Tumor volume was measured every week using a caliper, calculated as (length × width2)/2. After 4 weeks, mice were sacrificed and checked for final tumor weight.
item724 Twelve female BALB/c nude mice (aged 4 weeks, 18-22g) were randomly divided into 2 groups. Stable circMETTL3-expression SUM1315 cells or control cells (1×106 cells in 0.1 mL PBS) was subcutaneously injected into mammary fat pads of the mice and the growth of tumors was followed up every week. Tumor volume was measured every week using a caliper, calculated as (length × width2)/2. After 4 weeks, mice were sacrificed and checked for final tumor weight.
item725 Upon the development of leukemic disease (established using a white blood cell (WBC) count), 0.1 mg/kg or 0.5 mg/kg of SsD was intraperitoneally injected three times per week for three consecutive weeks.
item726 The 40 nude mice of each large group were classified into 8 small groups (n = 5) and subcutaneously injected with transfected cell suspension in the back (5 × 106 cells/mL/mouse). The length and width of tumors were recorded every 4 days, and the tumor volume = (length × width2)/2. The tumor growth curve was thereby graphed. On the 28th day of injection, mice were euthanized by neck dislocation, and the xenografts were harvested, photographed, and weighed
item727 The 40 nude mice of each large group were classified into 8 small groups (n = 5) and subcutaneously injected with transfected cell suspension in the back (5 × 106 cells/mL/mouse). The length and width of tumors were recorded every 4 days, and the tumor volume = (length × width2)/2. The tumor growth curve was thereby graphed. On the 28th day of injection, mice were euthanized by neck dislocation, and the xenografts were harvested, photographed, and weighed
item728 Hepatocyte-specific knockout USP48 was obtained by crossing Alb-Cre mice with USP48flox/flox mice.
item729 Hepatocyte-specific knockout USP48 was obtained by crossing Alb-Cre mice with USP48flox/flox mice.
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item733 U87 cells (5 × 105) transfected with an empty vector, METTL3 shRNA, or METTL3 overexpression vector were inoculated into the right frontal node of nude mice.
item734 The mice were maintained in cages under standard environmental conditions (temperature 25 ± 2 ℃, humidity 55 ± 5% and 12-h light/12-h dark cycle) and given free access to food and tap water. All mice were randomly divided into three groups with 6 in each group. To establish xenograft model, A549 cells (1 × 107) transfected with si-control (si-NC), si-ALKBH5 or si-ALKBH5 + RMRP were injected subcutaneously into a single side of the armpit of each mouse.
item735 HCT8/T xenografts derived from shNC or shIGF2BP3-1 HCT8/T cells were established through subcutaneous inoculation of cells (6×106) into nude mice.
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item738 U251/TR cells (2 × 106 per mouse) with stable transfection of sh-circ_0072083 or sh-NC were subcutaneously injected into mice.
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item743 For the ROS-induced DNA damage analysis, the indicated cell lines were treated with or without 100 uM hydrogen peroxide (H2O2), or 80 uM Carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 6 hours. For the in vivo ROS study, DMSO and 5 mg/kg CCCP was intraperitoneally injected in to three pairs of mice.
item744 For the ROS-induced DNA damage analysis, the indicated cell lines were treated with or without 100 uM hydrogen peroxide (H2O2), or 80 uM Carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 6 hours. For the in vivo ROS study, DMSO and 5 mg/kg CCCP was intraperitoneally injected in to three pairs of mice.
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item747 The mice were housed with filtered air, 12 h light/dark cycle, constant temperature (25℃), relative humidity (50±5%) and free access to food and water. In order to establish a human NSCLC xenograft model, 5×106 H1299, sh-METTL3-H1299 and METTL3 stably overexpressed H1299 cells (2×106 per mouse) were subcutaneously injected into mice. Tumor growth was observed daily. Tumor volume was calculated as follows: 0.5× (length × width2). At 24 days post-inoculation, the maximum diameter exhibited by a single subcutaneous tumor was 15 mm and mice were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg), then sacrificed by cervical dislocation.
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item751 Adult male C57BL/6J mice (weighting 18-25 g) were obtained from Laboratory Animal Center, Zhengzhou University. Mice were subcutaneously injected with 1 × 107 CEM-C1 cells for tumorigenesis and randomly divided into four group: control group; control + Dex; sh-RNA + Dex; sh-USP1 + Dex. Mice treatment with Dex were intraperitoneally injected with 8 mg/kg Dex every day for 10 consecutive days after tumor growth and mice treatment with sh-RNA or sh-USP1 were injected intravenously with 2 mg/Kg sh-RNA or USP1 sh-RNA. The control group of mice were injected with the same volume of normal saline. After the treatment of each group, the mice were housed and fed in a room with an ambient temperature of 25℃, and the survival time, weight of the mice, and tumor weight were recorded. When rats were sacrificed, tissues were harvested for Western blot analysis.
item752 Adult male C57BL/6J mice (weighting 18-25 g) were obtained from Laboratory Animal Center, Zhengzhou University. Mice were subcutaneously injected with 1 × 107 CEM-C1 cells for tumorigenesis and randomly divided into four group: control group; control + Dex; sh-RNA + Dex; sh-USP1 + Dex. Mice treatment with Dex were intraperitoneally injected with 8 mg/kg Dex every day for 10 consecutive days after tumor growth and mice treatment with sh-RNA or sh-USP1 were injected intravenously with 2 mg/Kg sh-RNA or USP1 sh-RNA. The control group of mice were injected with the same volume of normal saline. After the treatment of each group, the mice were housed and fed in a room with an ambient temperature of 25℃, and the survival time, weight of the mice, and tumor weight were recorded. When rats were sacrificed, tissues were harvested for Western blot analysis.
item753 Mice were provided adlibitum with water and a standard laboratory chow diet. The animal room was held a standard temperature, humidity and illumination. After intraperitoneal injection of FTO overexpression vector and NR to mice for 7 days, inguinal white adipose tissue (iWAT) were collected from mice.
item754 Mice were provided adlibitum with water and a standard laboratory chow diet. The animal room was held a standard temperature, humidity and illumination. After intraperitoneal injection of FTO overexpression vector and NR to mice for 7 days, inguinal white adipose tissue (iWAT) were collected from mice.
item755 To induce recombination at 8 weeks of age both CAG-CreERT;Ythdf2fl/fl and Ythdf2fl/fl littermates were injected with 75mg/kg body weight tamoxifen dissolved in corn oil daily for 5 days.
item756 Neonatal mouse cardiomyocytes (CMs) were blinded to isolate from 7-10 cases of postnatal 1-day old wildtype C57/B mice and cultured using Pierce Primary Cardi Page 10.omyocyte Isolation Kit (Thermo Fisher Scientific) as the manufacturer's instructions.
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item769 For the in vivo tumorigenicity assay, female BALB/c nude mice (ages 4-5 weeks) were randomly divided into two groups (n = 6/group). Calu1 cells (4 × 106) that had been stably transfected with sh-LCAT3 or scramble were implanted subcutaneously into the nude mice. Tumor growth was measured after one week, and tumor volumes were calculated with the following formula: Volume (cm3) = (length × width2)/2. After four weeks, the mice were euthanized, and the tumors were collected and weighed. For the in vivo tumor invasion assay, 1.2 × 106 scramble or shLCAT3 cells were injected intravenously into the tail vein of nude mice (n = 6/group). 1.5 mg luciferin (Gold Biotech, St Louis, MO, USA) was administered once a week for 4 weeks, to monitor metastases using an IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA).
item770 For the in vivo tumorigenicity assay, female BALB/c nude mice (ages 4-5 weeks) were randomly divided into two groups (n = 6/group). Calu1 cells (4 × 106) that had been stably transfected with sh-LCAT3 or scramble were implanted subcutaneously into the nude mice. Tumor growth was measured after one week, and tumor volumes were calculated with the following formula: Volume (cm3) = (length × width2)/2. After four weeks, the mice were euthanized, and the tumors were collected and weighed. For the in vivo tumor invasion assay, 1.2 × 106 scramble or shLCAT3 cells were injected intravenously into the tail vein of nude mice (n = 6/group). 1.5 mg luciferin (Gold Biotech, St Louis, MO, USA) was administered once a week for 4 weeks, to monitor metastases using an IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA).
item771 For the in vivo tumorigenicity assay, female BALB/c nude mice (ages 4-5 weeks) were randomly divided into two groups (n = 6/group). Calu1 cells (4 × 106) that had been stably transfected with sh-LCAT3 or scramble were implanted subcutaneously into the nude mice. Tumor growth was measured after one week, and tumor volumes were calculated with the following formula: Volume (cm3) = (length × width2)/2. After four weeks, the mice were euthanized, and the tumors were collected and weighed. For the in vivo tumor invasion assay, 1.2 × 106 scramble or shLCAT3 cells were injected intravenously into the tail vein of nude mice (n = 6/group). 1.5 mg luciferin (Gold Biotech, St Louis, MO, USA) was administered once a week for 4 weeks, to monitor metastases using an IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA).
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item779 Subcutaneously injected with 5 × 106 Jurkat cells and fed under Specific Pathogen Free (SPF) conditions. One week later, tumor mass was injected once a week with Agomir NC, Agomir-211, Antagomir NC and Antagomir 211, respectively, for three weeks.
item780 .
item781 IGF2BP2 activates the expression of ErbB2 by recognizing the m6A of YAP, thus affecting the cell cycle of CRC, inhibiting cell apoptosis, and promoting proliferation.
item782 IGF2BP2 activates the expression of ErbB2 by recognizing the m6A of YAP, thus affecting the cell cycle of CRC, inhibiting cell apoptosis, and promoting proliferation.
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item785 Subcutaneously injected shMETTL3 or shNC-expressing U87-MG-TMZ cells into BALB/c NOD mice. After confirmation of GBM implantation, mice were treated with TMZ (66 mg/kg/d, 5 d per week, for 3 cycles).
item786 Subcutaneously injected shMETTL3 or shNC-expressing U87-MG-TMZ cells into BALB/c NOD mice. After confirmation of GBM implantation, mice were treated with TMZ (66 mg/kg/d, 5 d per week, for 3 cycles).
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item817 For the PDX model, fresh patient HCC tissues were cut into fragments with a volume of 3 × 3 mm3 and then implanted subcutaneously into the flanks of nude mice. The mice were given sorafenib (30 mg/kg) or vehicle orally twice a week for 24 days. This procedure was approved by the Ethics Committee of Jinling Hospital.
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item819 CircARHGAP12-stable knockdown cervical cancer cells (100 uL PBS containing 5 × 106 cells) were subcutaneously injected into the lateral flank of BALB/c nude mice.
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item830 All mice were housed under a 12 h light/dark cycle with constant temperature about 25 ℃ and relative humidity approximating 55 %. The mice had free access to food and water for 10 days prior to the experiment. Forty mice were randomly selected and divided into four groups of 10 mice each. After 10 days, mice received an intraperitoneal injection of 22 mg / mL sodium pentobarbital (diluted in saline) followed by 167 uM LPS (60 uL) Saline solution was instilled into the oral cavity through the posterior pharyngeal wall. Pinch the nares quickly and hold for 30 s, model is successful when all fluid is absorbed into the nasal cavity, and slight tracheal rales appear. Lentiviral vectors containing pcDNA-SNHG4 (150 uM) or pcDNA-3.1 were intratracheally injected into mice. Twenty-one days after establishing the model, mice were intraperitoneally injected with 3% sodium pentobarbital and euthanized by overdose anesthesia at a dose of 90 mL/Kg, and organs and tissues were removed for follow-up studies.
item831 All mice were housed under a 12 h light/dark cycle with constant temperature about 25 ℃ and relative humidity approximating 55 %. The mice had free access to food and water for 10 days prior to the experiment. Forty mice were randomly selected and divided into four groups of 10 mice each. After 10 days, mice received an intraperitoneal injection of 22 mg / mL sodium pentobarbital (diluted in saline) followed by 167 uM LPS (60 uL) Saline solution was instilled into the oral cavity through the posterior pharyngeal wall. Pinch the nares quickly and hold for 30 s, model is successful when all fluid is absorbed into the nasal cavity, and slight tracheal rales appear. Lentiviral vectors containing pcDNA-SNHG4 (150 uM) or pcDNA-3.1 were intratracheally injected into mice. Twenty-one days after establishing the model, mice were intraperitoneally injected with 3% sodium pentobarbital and euthanized by overdose anesthesia at a dose of 90 mL/Kg, and organs and tissues were removed for follow-up studies.
item832 .
item833 Osmotic mini-pumps containing AngII (1 ug/kg/min, Enzo Bioche) were implanted in 7-week-old male mice. To interfere with the expression of KIAA1429, ALKBH5, or DDX6 in vivo, adeno-associated virus 9 (AAV9) vectors carrying a variety of overexpression plasmids or interfering RNA were randomly injected through the tail vein to C57BL/6N mice.
item834 Osmotic mini-pumps containing AngII (1 ug/kg/min, Enzo Bioche) were implanted in 7-week-old male mice. To interfere with the expression of KIAA1429, ALKBH5, or DDX6 in vivo, adeno-associated virus 9 (AAV9) vectors carrying a variety of overexpression plasmids or interfering RNA were randomly injected through the tail vein to C57BL/6N mice.
item835 BALB/cnu/nu mice (4-5 weeks old) were used for the xenograft experiment. The mice were randomly divided into 2 groups (n = 6 for each group) and injected with 5 × 106 HT-1197 cells in control group or FTO plasmid group, respectively.
item836 BALB/cnu/nu mice (4-5 weeks old) were used for the xenograft experiment. The mice were randomly divided into 2 groups (n = 6 for each group) and injected with 5 × 106 HT-1197 cells in control group or FTO plasmid group, respectively.
item837 BALB/cnu/nu mice (4-5 weeks old) were used for the xenograft experiment. The mice were randomly divided into 2 groups (n = 6 for each group) and injected with 5 × 106 HT-1197 cells in control group or FTO plasmid group, respectively.
item838 BALB/cnu/nu mice (4-5 weeks old) were used for the xenograft experiment. The mice were randomly divided into 2 groups (n = 6 for each group) and injected with 5 × 106 HT-1197 cells in control group or FTO plasmid group, respectively.
item839 BALB/cnu/nu mice (4-5 weeks old) were used for the xenograft experiment. The mice were randomly divided into 2 groups (n = 6 for each group) and injected with 5 × 106 HT-1197 cells in control group or FTO plasmid group, respectively.
item840 After adaptive feeding for 1 week, db/db mice were randomly divided into five groups (n = 6): db/db group, db/db + rAAV group, db/db + rAAV-METTL14 group, db/db + rAAV-klotho group, and db/db + rAAV-METTL14 + rAAV-klotho group. Except db/db group, the other four groups were injected with recombinant adeno-associated virus (rAAV) control, rAAV mediated delivery of METTL14 (rAAV-METTL14), or/and rAAV mediated delivery of klotho (rAAV-klotho) respectively via tail vein. Six db/m mice were chosen as the normal control.
item841 .
item842 To establish a tumour model, C57BL/6 mice were intraperitoneal injected with 25 mg/kg diethylnitrosamine at 2 weeks of age.
item843 To establish a tumour model, C57BL/6 mice were intraperitoneal injected with 25 mg/kg diethylnitrosamine at 2 weeks of age.
item844 To establish a tumour model, C57BL/6 mice were intraperitoneal injected with 25 mg/kg diethylnitrosamine at 2 weeks of age.
item845 Sixteen female goats in good body condition and suitable for pregnancy were selected. All selected goats underwent estrus synchronization treatment and were naturally mated. After 75 days of gestation, four male fetuses were removed from five pregnant goats during abortion operations, and their longissimus muscle samples were collected.
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item857 The growth of CC cells in vivo was examined by subcutaneous injection of LoVo cells or Caco-2 cells into NSG mice, while the metastatic ability of cells in vivo by intracardiac injection into mice.
item858 The number of cells inoculated in each mouse was 4 × 106, 1 × 106, 2 × 106 and 1 × 106, respectively.
item859 The number of cells inoculated in each mouse was 4 × 106, 1 × 106, 2 × 106 and 1 × 106, respectively.
item860 The number of cells inoculated in each mouse was 4 × 106, 1 × 106, 2 × 106 and 1 × 106, respectively.
item861 All mice were housed in specific pathogen-free conditions in the animal facility with constant temperature and humidity under a 12-h light/12-h dark cycle, with free access to water and food. After a week of adaptation, they were then divided into two groups randomly and fed with a HFD (60 kcal% fat, D12492, Research Diets) or standard normal chow diet (CD) for 16 weeks, respectively.
item862 All mice were housed in specific pathogen-free conditions in the animal facility with constant temperature and humidity under a 12-h light/12-h dark cycle, with free access to water and food. After a week of adaptation, they were then divided into two groups randomly and fed with a HFD (60 kcal% fat, D12492, Research Diets) or standard normal chow diet (CD) for 16 weeks, respectively.
item863 HCC-LM3 cells with silenced LNCAROD or the negative control were subcutaneously injected into the flank region of the mice at 2 × 106 cells.
item864 .
item865 The 60 female Kunming mice were divided into two groups (n = 30): control group (injection of physiological saline through the caudal vein) and colistin group (injection of 15 mg/kg colistin, twice a day, with an eight-hour interval).
item866 The 60 female Kunming mice were divided into two groups (n = 30): control group (injection of physiological saline through the caudal vein) and colistin group (injection of 15 mg/kg colistin, twice a day, with an eight-hour interval).
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item871 To establish mice model with ADR nephropathy, adult male C57BL/6J mice (8-12 weeks of age) were purchased from Animal Center of Fudan University and injected with 19.5 mg/kg ADR (D1515, Sigma-Aldrich, St-Louis, MO, USA) intravenously via tail vein.
item872 Used to inject 10 uL of the U87 MG-TMZ_R-luc cell suspension in the striatum at a depth of 3 mm from the dural surface. One week after the injection of the tumor cells, 40 mg/kg/day of TMZ in saline was administered for over 2 weeks by intraperitoneal injection.
item873 A total 5 × 106 stably transfected SiHa cells were subcutaneously injected into the flank of nude mice.
item874 .
item875 M3LKO (Mettl3fl/fl; Alb-Cre) mice were generated by crossing Mettl3fl/fl mice (provided by Dr. Jacob Hanna) with albumin-Cre mice (Jackson Laboratories, Bar Harbor, ME), and genotypes were confirmed by tail-DNA PCR using primers as previously described.
item876 M3LKO (Mettl3fl/fl; Alb-Cre) mice were generated by crossing Mettl3fl/fl mice (provided by Dr. Jacob Hanna) with albumin-Cre mice (Jackson Laboratories, Bar Harbor, ME), and genotypes were confirmed by tail-DNA PCR using primers as previously described.
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item880 VA-Lip-Mettl4-shRNA, VA-Lip-Fto-Plasmid and VA-Lip-Ythdf1-shRNA (0.75 mg/kg) were injected intravenously 3 times a week.
item881 .
item882 LUAD cells stably HCG11 and/or LATS1 overexpressed or silenced were subcutaneously injected into the flank of the BALB/c nude mice (male, 4 weeks old).
item883 LUAD cells stably HCG11 and/or LATS1 overexpressed or silenced were subcutaneously injected into the flank of the BALB/c nude mice (male, 4 weeks old).
item884 .
item885 Four-week-old male BALB/c nude mice (purchased from Lingchang company) were randomly divided into three groups, each group has five mice. Each of the mice was injected subcutaneously on the right lateral back with 1 × 106 of each lentivirus infected SW480 cells in which hnRNPA2B1 was knocked out or negative control cells. Mice were killed at day 29, and tumors were then isolated, photographed.
item886 Four-week-old male BALB/c nude mice (purchased from Lingchang company) were randomly divided into three groups, each group has five mice. Each of the mice was injected subcutaneously on the right lateral back with 1 × 106 of each lentivirus infected SW480 cells in which hnRNPA2B1 was knocked out or negative control cells. Mice were killed at day 29, and tumors were then isolated, photographed.
item887 A498 cells (1 × 106 cells) were resuspended in 100 uL of PBS and subcutaneously injected into the axillary fossa of nude mice (BALB/c-nude, 4 weeks old).
item888 .
item889 The WT-si-NC, WT-si-Ythdc1 and WT-si-Sqstm1 groups were intracutaneously injected with corresponding siRNAs (si-NC, si-Ythdc1, or si-Sqstm1, 2.5 nmol) on the circle.
item890 The WT-si-NC, WT-si-Ythdc1 and WT-si-Sqstm1 groups were intracutaneously injected with corresponding siRNAs (si-NC, si-Ythdc1, or si-Sqstm1, 2.5 nmol) on the circle.
item891 .
item892 A total of 2×106 tumor cells were suspended in 200 uL of PBS and injected the right flank of nude mice. The tumor sizes were measured weekly as soon as the tumors were measurable, and the tumor volumes were calculated using the following formula: volume (mm3) = width2 (mm2) × length (mm)/2. After 4 weeks, the mice were sacrificed, and the tumors were harvested.
item893 Indicated cells (1 × 107) were subcutaneously injected into 4-week-old male nude mice. Tumor volume was measured every 5 days.
item894 Indicated cells (1 × 107) were subcutaneously injected into 4-week-old male nude mice. Tumor volume was measured every 5 days.
item895 .
item896 The knockout first allele was converted into a conditional allele by crossing Ythdf2 knockout first mice with Flp deleter mice. The resulting floxed Ythdf2 mice were crossed with Vasa-GFPCre knock-in mice to allow specific knockout of Ythdf2 in germ cells.
item897 Male C57BL/6 mice (4-6 weeks old) were used in all tumor allografting experiments and transplanted with GL261-luc cells (1×105) into the frontal lobes of brains.
item898 .
item899 .
item900 .
item901 All mice described above were maintained on the C57BL/6 J background. Mice lacking Mettl3 in oocytes (referred to as Mettl3Gdf9 cKO) were generated by crossing Mettl3flox/flox mice with Gdf9-Cre mice. The Mettl3flox/flox female mice were used as the control group (referred to as WT). For the fertility test, six pairs of 6 weeks Mettl3flox/flox and Mettl3Gdf9 cKO female mice were randomly selected and continually mated to Mettl3flox/flox male mice which have been confirmed fertility for 5 months. The number of pups and litter size from each female was recorded.
item902 All mice described above were maintained on the C57BL/6 J background. Mice lacking Mettl3 in oocytes (referred to as Mettl3Gdf9 cKO) were generated by crossing Mettl3flox/flox mice with Gdf9-Cre mice. The Mettl3flox/flox female mice were used as the control group (referred to as WT). For the fertility test, six pairs of 6 weeks Mettl3flox/flox and Mettl3Gdf9 cKO female mice were randomly selected and continually mated to Mettl3flox/flox male mice which have been confirmed fertility for 5 months. The number of pups and litter size from each female was recorded.
item903 Chose 4-week-old female BALB/c nude mice for tumor xenograft experiments, which randomly were divided into four groups (n = 5 per group). Bladder cancer cells (3 × 106) were subcutaneously injected into the right axilla of the nude mice.
item904 Chose 4-week-old female BALB/c nude mice for tumor xenograft experiments, which randomly were divided into four groups (n = 5 per group). Bladder cancer cells (3 × 106) were subcutaneously injected into the right axilla of the nude mice.
item905 Chose 4-week-old female BALB/c nude mice for tumor xenograft experiments, which randomly were divided into four groups (n = 5 per group). Bladder cancer cells (3 × 106) were subcutaneously injected into the right axilla of the nude mice.
item906 To create the xenograft neoplasm system, 40 male BALB/c nude mice aged 5 weeks were randomly separated into sh-NC, sh-circHPS5, sh-circHPS5+CTRL, and sh-circHPS5+SAH groups (n = 5 for each group). HCC cells were subcutaneously injected into the axilla of the nude mice.
item907 To create the xenograft neoplasm system, 40 male BALB/c nude mice aged 5 weeks were randomly separated into sh-NC, sh-circHPS5, sh-circHPS5+CTRL, and sh-circHPS5+SAH groups (n = 5 for each group). HCC cells were subcutaneously injected into the axilla of the nude mice.
item908 To create the xenograft neoplasm system, 40 male BALB/c nude mice aged 5 weeks were randomly separated into sh-NC, sh-circHPS5, sh-circHPS5+CTRL, and sh-circHPS5+SAH groups (n = 5 for each group). HCC cells were subcutaneously injected into the axilla of the nude mice.
item909 To create the xenograft neoplasm system, 40 male BALB/c nude mice aged 5 weeks were randomly separated into sh-NC, sh-circHPS5, sh-circHPS5+CTRL, and sh-circHPS5+SAH groups (n = 5 for each group). HCC cells were subcutaneously injected into the axilla of the nude mice.
item910 To create the xenograft neoplasm system, 40 male BALB/c nude mice aged 5 weeks were randomly separated into sh-NC, sh-circHPS5, sh-circHPS5+CTRL, and sh-circHPS5+SAH groups (n = 5 for each group). HCC cells were subcutaneously injected into the axilla of the nude mice.
item911 To create the xenograft neoplasm system, 40 male BALB/c nude mice aged 5 weeks were randomly separated into sh-NC, sh-circHPS5, sh-circHPS5+CTRL, and sh-circHPS5+SAH groups (n = 5 for each group). HCC cells were subcutaneously injected into the axilla of the nude mice.
item912 Mice were anaesthetized with isoflurane supplied in a mouse anaesthesia apparatus, followed with joint surgery on the right joint by sectioning the medial meniscotibial ligament.
item913 Mice were anaesthetized with isoflurane supplied in a mouse anaesthesia apparatus, followed with joint surgery on the right joint by sectioning the medial meniscotibial ligament.
item914 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item915 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item916 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item917 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item918 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item919 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item920 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item921 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item922 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item923 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item924 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item925 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item926 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item927 After being anesthetized with urethane (i.p.), SD rats were endotracheally intubated and ventilated using an animal ventilator under the conditions: respiratory rate of 70 breaths/min, tidal volume of 20 ml/kg, and inspiratory/expiratory ratio of 1:1.
item928 .
item929 Approximately 1 × 107 stably transfected T24 cells were subcutaneously injected into BALB/c nude mice. The length (L) and width (W) of the tumours were measured weekly using callipers, while their volume was calculated using the equation: V = (L × W2)/2. After 4 weeks of injections, the mice were euthanised, and the tumour tissues were removed and weighed.
item930 Approximately 1 × 107 stably transfected T24 cells were subcutaneously injected into BALB/c nude mice. The length (L) and width (W) of the tumours were measured weekly using callipers, while their volume was calculated using the equation: V = (L × W2)/2. After 4 weeks of injections, the mice were euthanised, and the tumour tissues were removed and weighed.
item931 .
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item940 .
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item947 .
item948 The 8-10 weeks old mice were fed either a high fat diet or HF-CDAA , ad lib for 6-12 weeks. Chow diet was used as control for HFD.The mouse liver was perfused with PBS through portal vein, and liver tissue was cut into small pieces by a scissor. The single cell was made using syringe plunger to mull the tissue, and passed through a 40 uM cell strainer.
item949 The 8-10 weeks old mice were fed either a high fat diet or HF-CDAA , ad lib for 6-12 weeks. Chow diet was used as control for HFD.The mouse liver was perfused with PBS through portal vein, and liver tissue was cut into small pieces by a scissor. The single cell was made using syringe plunger to mull the tissue, and passed through a 40 uM cell strainer.
item950 The 8-10 weeks old mice were fed either a high fat diet or HF-CDAA , ad lib for 6-12 weeks. Chow diet was used as control for HFD.The mouse liver was perfused with PBS through portal vein, and liver tissue was cut into small pieces by a scissor. The single cell was made using syringe plunger to mull the tissue, and passed through a 40 uM cell strainer.
item951 The 8-10 weeks old mice were fed either a high fat diet or HF-CDAA , ad lib for 6-12 weeks. Chow diet was used as control for HFD.The mouse liver was perfused with PBS through portal vein, and liver tissue was cut into small pieces by a scissor. The single cell was made using syringe plunger to mull the tissue, and passed through a 40 uM cell strainer.
item952 The 8-10 weeks old mice were fed either a high fat diet or HF-CDAA , ad lib for 6-12 weeks. Chow diet was used as control for HFD.The mouse liver was perfused with PBS through portal vein, and liver tissue was cut into small pieces by a scissor. The single cell was made using syringe plunger to mull the tissue, and passed through a 40 uM cell strainer.
item953 The 8-10 weeks old mice were fed either a high fat diet or HF-CDAA , ad lib for 6-12 weeks. Chow diet was used as control for HFD.The mouse liver was perfused with PBS through portal vein, and liver tissue was cut into small pieces by a scissor. The single cell was made using syringe plunger to mull the tissue, and passed through a 40 uM cell strainer.
item954 5 × 105 selected cells were injected via the tail vein into 4- to 5-week-old NCG mice.
item955 .
item956 .
item957 .
item958 .
item959 Mouse subcutaneous xenograft and lung metastasis experiments were carried out with six 4-week-old male BALB/c nude mice.
item960 Mouse subcutaneous xenograft and lung metastasis experiments were carried out with six 4-week-old male BALB/c nude mice.
item961 .
item962 .
item963 .
item964 .
item965 .
item966 .
item967 .
item968 .
item969 1 × 106 (100 ul) cells of infected and uninfected by lentiviral were, respectively, injected subcutaneously into nude mice which divided randomly into scramble group and shKCNQ1OT1-1 group.
item970 1 × 106 (100 ul) cells of infected and uninfected by lentiviral were, respectively, injected subcutaneously into nude mice which divided randomly into scramble group and shKCNQ1OT1-1 group.
item971 .
item972 .
item973 .
item974 .
item975 .
item976 The U87MG cells (1 × 107 cells in 0.1 ml PBS) were injected subcutaneously into BALB/c nude mice. Tumor width and length were recorded every 5 days.
item977 The mice were maintained on a 12-h light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.). On day 7.5 of pregnancy (E7.5), ethionine (Sigma-Aldrich, USA) was intraperitoneally injected only once at a dose of 500 mg/kg to establish the NTDs embryo model. And SAM (MedChemExpress, USA) was intraperitoneally injected only once at a dose of 30 mg/kg. The same dose was intraperitoneally injected to the pregnant mice for control group.
item978 The mice were maintained on a 12-h light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.). On day 7.5 of pregnancy (E7.5), ethionine (Sigma-Aldrich, USA) was intraperitoneally injected only once at a dose of 500 mg/kg to establish the NTDs embryo model. And SAM (MedChemExpress, USA) was intraperitoneally injected only once at a dose of 30 mg/kg. The same dose was intraperitoneally injected to the pregnant mice for control group.
item979 .
item980 Groups of HCT116-Luc-shCtrl, HCT116-Luc-shLINC00460, and HCT116-Luc-shLINC00460 + HMGA1 cells (5 × 106) were injected subcutaneously into the flanks of mice correspondingly.
item981 Groups of HCT116-Luc-shCtrl, HCT116-Luc-shLINC00460, and HCT116-Luc-shLINC00460 + HMGA1 cells (5 × 106) were injected subcutaneously into the flanks of mice correspondingly.
item982 Specific pathogen-free (SPF) female NOD/SCID mice (5-6 weeks old) were randomly distributed into two groups: the OECtrl group and the OEMETTL14 groups. Phosphate buffer (200 uL) containing approximately 5 × 107 HSC3 or CAL33 cells was subcutaneously injected into the inner thigh of each mouse. The mice were euthanized two weeks after injection, and the tumour xenografts were harvested, photographed, weighed, and fixed.
item983 For the subcutaneous implantation model, 5×106 control and CASC11 overexpressing Huh7 cells were suspended in the 100 uL serum-free DMEM medium and then injected subcutaneously. For the metastatic model, 1×105 control and CASC11 overexpressing Huh7 cells were suspended in the 50 uL serum-free DMEM medium, followed by being injected into the tail vein of nude mice.
item984 For the subcutaneous implantation model, 5×106 control and CASC11 overexpressing Huh7 cells were suspended in the 100 uL serum-free DMEM medium and then injected subcutaneously. For the metastatic model, 1×105 control and CASC11 overexpressing Huh7 cells were suspended in the 50 uL serum-free DMEM medium, followed by being injected into the tail vein of nude mice.
item985 .
item986 Clone C4 was expanded and electroporated with Cre-expressing plasmid pOG231, followed by culture in media containing 2 uM ganciclovir. Ninety-two clones were screened for removal of the HyTK cassette and presence of loxP sites flanking Ythdc2 exons 13-16, resulting in nine positive clones (Ythdc2fl/+).
item987 Male Sprague-Dawley rats (375-400 g) liver fibrosis was induced by subcutaneous injection of carbon tetrachloride (CCl4) and olive oil (a ratio of 2:3) twice per week.
item988 Mice were inoculated subcutaneously with 5× 106 corresponding cells per flank in 150 uL of 50% Matrigel Matrix (Corning). Tumor diameters were measured semi-weekly with a caliper and tumor volumes calculated using the formula: (width2 × length)/2 = V (mm3).
item989 NOD.CB17-Prkdcscid/J none castrated SCID male mice 4 weeks old were purchased from Jackson Laboratories. After a one week period of acclimation, the mice were injected with 6×106 TetOn-Flag-ELF3 LNCaP suspended in 200 uL of a 50% mixture containing RPMI 1640 medium and Matrigel matrix basement membrane (BD Corporation, Bedford, MA) subcutaneously into the right flank region. The mice were either fed with doxycycline 200 mg/kg containing diet (BioServ, NJ) to induce the ELF3 expression or with a control regular diet. The sizes of the resultant tumors were measured weekly.
item990 In the present study, we assessed Smad3 +/- mice for a comparatively long period to examine the effects of Smad3 expression and phosphorylation and to evaluate the involvement of Smad3 in DN. Heterozygous Smad3- knockout mice were kindly provided by Dr. Yasue, University of Tokushima. We attempted to generate Smad3-null mice using pairs of Smad3 mice, but progeny was rarely obtained, and pups were fragile and could not survive for a long period (5 weeks at the most). Therefore, BKS/Cg-m Lepr db (db/db) x Smad3 +/- mice were developed using pairs of Lepr db +/- x Smad3 +/-. Smad3 +/-;db/+ mice were generated by crossing Smad3 +/- and db/+ mice. Moreover, Smad3 +/-;db/db were generated by crossing Smad3 +/-;db/+ and db/+ mice as previously described. Blood glucose concentrations were measured from the tail vein (glucose assay kit; Abbott). The diabetic phenotype was confirmed 4 weeks after birth by blood glucose >300 mg/dl.
item991 .
item992 .
item993 The male BALB/c nude mice were randomized divide into two groups, each group including six 4 weeks old nude mice. Investigators were blinded to the treatment groups during data collection and subsequent data analysis. In the subcutaneous xenograft model, 5 × 105 cells were subcutaneously injected in the right flanks of nude mice. In the orthotopic intracranial mouse model, each mouse was intracranially injected with 1 × 105 luciferase transfected U87MG cells in 10 uL PBS solution.
item994 The male BALB/c nude mice were randomized divide into two groups, each group including six 4 weeks old nude mice. Investigators were blinded to the treatment groups during data collection and subsequent data analysis. In the subcutaneous xenograft model, 5 × 105 cells were subcutaneously injected in the right flanks of nude mice. In the orthotopic intracranial mouse model, each mouse was intracranially injected with 1 × 105 luciferase transfected U87MG cells in 10 uL PBS solution.
item995 The male BALB/c nude mice were randomized divide into two groups, each group including six 4 weeks old nude mice. Investigators were blinded to the treatment groups during data collection and subsequent data analysis. In the subcutaneous xenograft model, 5 × 105 cells were subcutaneously injected in the right flanks of nude mice. In the orthotopic intracranial mouse model, each mouse was intracranially injected with 1 × 105 luciferase transfected U87MG cells in 10 uL PBS solution.
item996 The male BALB/c nude mice were randomized divide into two groups, each group including six 4 weeks old nude mice. Investigators were blinded to the treatment groups during data collection and subsequent data analysis. In the subcutaneous xenograft model, 5 × 105 cells were subcutaneously injected in the right flanks of nude mice. In the orthotopic intracranial mouse model, each mouse was intracranially injected with 1 × 105 luciferase transfected U87MG cells in 10 uL PBS solution.
item997 .
item998 .
item999 .
item1000 Severe combined immunodeficiency (SCID) female mice aged 6-8 weeks were transplanted subcutaneously with 2 × 107 P4-pBIG2i-hIGFBP3 or P4-pBIG2i transfectant cells. Tumor growth was measured using Vernier calipers and the volume was calculated using the formula: length × width × width × 0.52, which approximates the volume of an elliptical solid mass. Doxycycline at 2 mg/mL in drinking water with 2% sucrose was used to feed the animals when the tumor size was over 0.5 cm in diameter. The xenograft tumors were removed from five animals from each group on days 4, 7, 11, 14, and 20 after doxycycline treatment.
item1001 Implanting 5000 human derived GSCs into the right cerebral cortex of NSG mice at a depth of 3.5 mm under a University of California, San Diego Institutional Animal Care and Use Committee (IACUC) approved protocol. Brains were harvested and fixed in 4% formaldehyde, cryopreserved in 30% sucrose, and then cryosectioned. Hematoxylin and eosin (H&E) staining was performed on sections for histological analysis. In parallel survival experiments, mice were observed until the development of neurological signs. For in vivo drug treatment studies, intracranial xenografts were generated by implanting 5000 patient-derived GSCs (387 and 4121) into the right cerebral cortex of NSG mice as described above. Mice recovered for 7 days were randomly assigned into drug vs. treatment group by a blinded investigator. Mice were then treated daily with either vehicle (25 mM Tartaric acid) or 50 mg/kg linsitinib by oral gavage.
item1002 Paired littermates of F2 (Igfbp3+/+:KrasG12D/+ and Igfbp3-/-:KrasG12D/+) were sacrificed ranging from ages 4 to 7 months. After preliminary analysis of F2 mice, we sacrificed 5-month-old Igfbp3+/+:KrasG12D/+ and Igfbp3-/-KrasG12D/+ mice that had been backcrossed to S129 background for representative analysis. The lung tissue was immediately removed after the mice were sacrificed and visible pleural nodules were counted directly.
item1003 .
item1004 3 to 5-week old female BALB/c athymic (NU/NU) nude mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into indicated groups (five mice per group). To assess the in vivo effects of cycloleucine, the xenografted tumors had reached approximately 5 mm in diameter from mice and then these xenografted mice were feed with Vehicle or cycloleucine (25 mg/kg twice weekly) and tumor volume were measured every 3 day.
item1005 .
item1006 .
item1007 .
item1008 BALB/c nude mice (5 weeks old) were purchased from the Beijing HFK Bioscience Co. Ltd. 2×106 cells (50 uL) were mixed with 50 uL Matrigel (BD Biosciences, San Jose, CA, USA) were injected subcutaneously in the rear flank fat pad of the nude mice (N = 6, per group).
item1009 .
item1010 .
item1011 HepG2 wild-type, KO1# 1 and KO1# 2 cells (about 5 × 106) were used to establish subcutaneous (SC) xenograft tumor models. Tumor size was monitored weekly. Mice were sacrificed after 4 weeks, and tumor weight and two dimensions of tumors were measured.
item1012 2 × 105 dissociated cells in 2 uL PBS were injected into the following site (anteroposterior [AP] +0.6 mm, mediolateral [ML] +1.6 mm, and dorsoventricular [DV] 2.6 mm) with a rate of 1 uL/min.
item1013 2 × 105 dissociated cells in 2 uL PBS were injected into the following site (anteroposterior [AP] +0.6 mm, mediolateral [ML] +1.6 mm, and dorsoventricular [DV] 2.6 mm) with a rate of 1 uL/min.
item1014 For high-fat-diet treatment, 3-week-old male mice were randomly divided into three groups: two experimental groups freely fed with HFD of 60% fat, 20% carbohydrate, and 20% protein (Cat#: D12492, Research Diets) or KD of 89.5% fat, 0.1% carbohydrate, and 10.4% protein (Cat#: D12369B, Research Diets), and the control group fed with SD of 10% fat, 70% carbohydrate, and 20% protein (Cat#: D12450B, Research Diets), respectively. The mice were allowed free access to water and food.For fasting treatment, 3-month-old male mice were randomly divided into two groups: the fasting group with only free access to water for 48 h and the control group allowed free access to water and SD.
item1015 .
item1016 .
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item1018 .
item1019 The NSGS mice were bred and subjected to the xeno-transplantation model. For the AML mouse model, 0.2 × 106 MONOMAC6 cells were directly transplanted into NSGS mice via tail vein. After 10 days, FB23-2 (2 mg/kg/day) and DMSO vehicle were intraperitoneally injected into the mice for a continuous 10 days. The mice were euthanized by CO2 inhalation if they exhibited classical AML symptoms including hunched posture, paralysis, and reduced body weight. Meanwhile, the PB, spleen, and liver samples were collected for further analysis.
item1020 The NSGS mice were bred and subjected to the xeno-transplantation model. For the AML mouse model, 0.2 × 106 MONOMAC6 cells were directly transplanted into NSGS mice via tail vein. After 10 days, FB23-2 (2 mg/kg/day) and DMSO vehicle were intraperitoneally injected into the mice for a continuous 10 days. The mice were euthanized by CO2 inhalation if they exhibited classical AML symptoms including hunched posture, paralysis, and reduced body weight. Meanwhile, the PB, spleen, and liver samples were collected for further analysis.
item1021 .
item1022 The mice were fed a normal chow diet (Harlan Teklad) and maintained in a standard 12-hour light/12-hour dark cycle. At the age of 10 weeks, both male and female mice were intraperitoneally injected with 250 L (20 mg/mL) tamoxifen every other day for three times to delete Mettl14 in Beta-cells. Intraperitoneal glucose tolerance tests were performed on mice at the age of 15 weeks after a 5-hour fast (2 g/kg dextrose). Insulin levels were measured at 0, 15, and 30 minutes after glucose challenge by using the Ultra Sensitive Mouse Insulin ELISA Kit .Insulin tolerance tests were performed after a 5-hour fast by administering human recombinant insulin (0.75 U/kg).
item1023 A number of 5 × 106 SMMC7721 or MHCC97H cells re-suspended in 100 uL of PBS were subcutaneously injected into the right flank of 6-week old male NCG mice.
item1024 A number of 5 × 106 SMMC7721 or MHCC97H cells re-suspended in 100 uL of PBS were subcutaneously injected into the right flank of 6-week old male NCG mice.
item1025 Exposed Sprague-Dawley rats to 0, 250, and 500 mg DEHP per kg body weight per day at the prepuberty stage from postnatal day 22 (PND 22) to PND 35 by oral gavage.
item1026 Exposed Sprague-Dawley rats to 0, 250, and 500 mg DEHP per kg body weight per day at the prepuberty stage from postnatal day 22 (PND 22) to PND 35 by oral gavage.
item1027 .
item1028 Thirty-two C57BL/6J male mice were randomly assigned to a CON (basal diet), RES (basal diet + 500 mg/kg resveratrol), AFB1 (basal diet + 600 ug/kg aflatoxin B1), and ARE (basal diet + 500 mg/kg resveratrol and 600 ug/kg aflatoxin B1) group for 4 weeks of feeding (n = 8/group). Briefly, redox status, apoptosis, and m6A modification in the liver were assessed. Compared to the CON group, the AFB1 group showed increased activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), prevalent vacuolization and cell edema, abnormal redox status, imbalance apoptosis, and especially, the higher expression of cleaved-caspase-3 protein.
item1029 Thirty-two C57BL/6J male mice were randomly assigned to a CON (basal diet), RES (basal diet + 500 mg/kg resveratrol), AFB1 (basal diet + 600 ug/kg aflatoxin B1), and ARE (basal diet + 500 mg/kg resveratrol and 600 ug/kg aflatoxin B1) group for 4 weeks of feeding (n = 8/group). Briefly, redox status, apoptosis, and m6A modification in the liver were assessed. Compared to the CON group, the AFB1 group showed increased activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), prevalent vacuolization and cell edema, abnormal redox status, imbalance apoptosis, and especially, the higher expression of cleaved-caspase-3 protein.
item1030 .
item1031 Zebrafish (Danio rerio) AB strain-derived Tg(lfabp:Dendra2-NTR)cq1 was used as WT, and rbm15cq96 mutant was generated by ENU treatment.
item1032 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1033 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1034 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1035 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1036 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1037 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1038 For each experiment, 6- to 8-week-old mice were used and randomly allocated to each group. For xenograft mouse, 0.1 × 106 MA9.3ITD cells were transplanted into NRGS recipient mice intravenously. Drug treatment was started from 10 days after transplantation. CS2 was administered through intraperitoneal (i.p.) injection at 5mg/kg/day, every other day. CS1 dissolved in saturated Beta-cyclodextrin (C0926, Sigma-Aldrich) solution was delivered by intravenous injection (i.v.). Successful engraftment was observed following 4 weeks post inoculation displaying a level of about 5% human CD33+ cells in peripheral. To generate PDX mouse models, 1 × 106 AML patient derived BMMNCs were transplanted into NRGS recipient mice intravenously, and drug treatment was started from 7 days later. CS2, FB23-2, and free CS1 were administered through i.p. injection at 5 mg/kg/day, while Micelle (900661, Sigma-Aldrich) packaged CS1 was delivered by i.v. injection at 5mg/kg/day. Both CS1 and CS2 were injected every other day for a total of ten times.
item1039 .
item1040 .
item1041 .
item1042 For in vivo drug treatment studies, intracranial xenografts were generated by implanting 5000 patient-derived GSCs (387 and 4121) into the right cerebral cortex of NSG mice as described above.
item1043 For in vivo drug treatment studies, intracranial xenografts were generated by implanting 5000 patient-derived GSCs (387 and 4121) into the right cerebral cortex of NSG mice as described above.
item1044 To establish a subcutaneous tumour model in nude mice, 2 × 107 Y79 cells (METTL3 knockdown group: shNC, shRNA1 and shRNA2; METTL3 up-regulated group: NC and METLL3) were resuspended in 1 mL of pre-cooled PBS, and 200 uL of the cell suspension was injected subcutaneously into the left side of the armpit to investigate tumour growth (4 × 106 per mouse).
item1045 To establish a subcutaneous tumour model in nude mice, 2 × 107 Y79 cells (METTL3 knockdown group: shNC, shRNA1 and shRNA2; METTL3 up-regulated group: NC and METLL3) were resuspended in 1 mL of pre-cooled PBS, and 200 uL of the cell suspension was injected subcutaneously into the left side of the armpit to investigate tumour growth (4 × 106 per mouse).
item1046 To establish a subcutaneous tumour model in nude mice, 2 × 107 Y79 cells (METTL3 knockdown group: shNC, shRNA1 and shRNA2; METTL3 up-regulated group: NC and METLL3) were resuspended in 1 mL of pre-cooled PBS, and 200 uL of the cell suspension was injected subcutaneously into the left side of the armpit to investigate tumour growth (4 × 106 per mouse).
item1047 To establish a subcutaneous tumour model in nude mice, 2 × 107 Y79 cells (METTL3 knockdown group: shNC, shRNA1 and shRNA2; METTL3 up-regulated group: NC and METLL3) were resuspended in 1 mL of pre-cooled PBS, and 200 uL of the cell suspension was injected subcutaneously into the left side of the armpit to investigate tumour growth (4 × 106 per mouse).
item1048 To establish a subcutaneous tumour model in nude mice, 2 × 107 Y79 cells (METTL3 knockdown group: shNC, shRNA1 and shRNA2; METTL3 up-regulated group: NC and METLL3) were resuspended in 1 mL of pre-cooled PBS, and 200 uL of the cell suspension was injected subcutaneously into the left side of the armpit to investigate tumour growth (4 × 106 per mouse).
item1049 Cell suspension of SW48 or SW48/TP53 (5-7 passages, 1 × 106 cells in 20 uL/mouse) was subcutaneously injected into the left flank of the mice.
item1050 .
item1051 .
item1052 .
item1053 .
item1054 .
item1055 .
item1056 .
item1057 .
item1058 .
item1059 .
item1060 .
item1061 .
item1062 Left anterior descending coronary artery (LAD) was ligated for 20 minutes, followed by 48 h reperfusion. Controls underwent same procedures except LAD ligation. WTAP shRNA vector or its negative control (shNC) was injected into the left ventricular anterior wall 24 h before I/R. A pressure volume catheter was used for cardiac function assay.
item1063 6- to 10-week-old female Rosa26Cas9/+ mice were treated daily for two weeks with either vehicle or 50 mg kg-1 STM2457 (STORM). Four weeks after treatment, bone marrow cells from these mice were freshly dissected (as mentioned above) and blocked with anti-mouse CD16/32 (BD Pharmigen, cat. no. 553142) and 10% mouse serum (Sigma).
item1064 .
item1065 .
item1066 .
item1067 6- to 12-week-old mice were used for experiments.
item1068 .
item1069 .
item1070 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1071 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1072 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1073 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1074 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1075 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1076 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1077 For MIA + SAH control, S-adenosylhomocysteine (SAH), Mettl3 inhibitor (10 mg/kg) (MCE, NJ, USA) was injected intraperitoneally before MIA injection and maintained twice a week until mice were sacrificed.
item1078 5 × 105 HCT116 CSCs were subcutaneously injected into the mice similarly as nude mice. Seven days later, 5, 10, or 20 mg/kg Berberine was intraperitoneally injected. The same volume of PBS was injected and considered as negative control.
item1079 Wild-type C57 (female, 12-16 weeks old), ALKBH5 /- mice (female and male, 12-16 weeks old), and SPF-grade SD rats (female, 180-230 g) were used to establish the AMI model.Sodium pentobarbital diluted to 10 mg/mL was used to anesthetize the mice or rat at the dose of 50 mg/kg through an intraperitoneal injection. By using a small animal ventilator with endotracheal intubation, thoracotomy was performed at the left fourth intercostal region. The heart was exposed, and the left anterior descending coronary artery (LCA) was occluded through a 6-0 silk suture that was placed 2-3 mm distal to the origin of the LCA with a slipknot. The apical region turned white, and ST segment elevation and T wave inversion of ECG showed that the AMI model was successfully established. Forty-five minutes after ischemia, the slipknot was released, and the ischemic region was reperfused. PBS (0.2 ml), HSSS (23.5 mg/kg, 0.2 ml), IOX1 (10 mg/kg, 0.2 ml), and HSSS-I (33.5 mg/kg, containing 10 mg/kg IOX1, 0.2 ml) were administered through caudal vein injection for 14 days at the frequency of one time per day.
item1080 Wild-type C57 (female, 12-16 weeks old), ALKBH5 /- mice (female and male, 12-16 weeks old), and SPF-grade SD rats (female, 180-230 g) were used to establish the AMI model.Sodium pentobarbital diluted to 10 mg/mL was used to anesthetize the mice or rat at the dose of 50 mg/kg through an intraperitoneal injection. By using a small animal ventilator with endotracheal intubation, thoracotomy was performed at the left fourth intercostal region. The heart was exposed, and the left anterior descending coronary artery (LCA) was occluded through a 6-0 silk suture that was placed 2-3 mm distal to the origin of the LCA with a slipknot. The apical region turned white, and ST segment elevation and T wave inversion of ECG showed that the AMI model was successfully established. Forty-five minutes after ischemia, the slipknot was released, and the ischemic region was reperfused. PBS (0.2 ml), HSSS (23.5 mg/kg, 0.2 ml), IOX1 (10 mg/kg, 0.2 ml), and HSSS-I (33.5 mg/kg, containing 10 mg/kg IOX1, 0.2 ml) were administered through caudal vein injection for 14 days at the frequency of one time per day.
item1081 The immunoprecipitated RNA samples from sham and 12h reperfusion groups (n = 5 each) were labeled with Cy5 fluorescent dye using Super RNA Labeling Kit (Arraystar) and purified using RNeasy Mini Kit. Cy5 labeled cRNAs were fragmented and hybridized to a mouse m6A epitranscriptomic microarray (8×60K, Arraystar) that contained 44,122 mRNA and 12,496 lncRNA degenerate probes. The hybridized arrays were scanned using an Agilent Scanner G2505C. For each of the 2 groups, 5 microarrays were used.
item1082 SMC-specific TWIST1-deficient mice were generated by crossing TWIST1flox/flox mice with animals expressing Sm22-Cre (smooth muscle protein 22-Cre). Deletion of TWIST1 in SMCs was confirmed by histology and Western blot.
item1083 .
item1084 The thoracic cavity of rats was exposed, and the left anterior descending coronary artery was ligated with a 6-0 silk thread. Successfully surgical MI could be observed, with myocardium color fading and pulse weakening. After 30 min of ischemia, the blood flow was restored by releasing the slipknot, and then 120-min perfusion was performed. Afterward, the thoracic cavity of rats was sutured. The rats were assigned into 4 groups, with 12 rats in each group. Lentivirus packaged short hairpin (sh)-negative control (NC) and sh-METTL3 (GenePharma, Shanghai, China) were injected into the rats via tail vein 24 h before operation. The titer of lentivirus was 1?×?109 TU/mL, and the injection rate was 0.2 ul/min for 10 min. Blood samples were collected 24 h after reperfusion.
item1085 The thoracic cavity of rats was exposed, and the left anterior descending coronary artery was ligated with a 6-0 silk thread. Successfully surgical MI could be observed, with myocardium color fading and pulse weakening. After 30 min of ischemia, the blood flow was restored by releasing the slipknot, and then 120-min perfusion was performed. Afterward, the thoracic cavity of rats was sutured. The rats were assigned into 4 groups, with 12 rats in each group. Lentivirus packaged short hairpin (sh)-negative control (NC) and sh-METTL3 (GenePharma, Shanghai, China) were injected into the rats via tail vein 24 h before operation. The titer of lentivirus was 1?×?109 TU/mL, and the injection rate was 0.2 ul/min for 10 min. Blood samples were collected 24 h after reperfusion.
item1086 .
item1087 About 5 × 106 MKN45 cells stably transfected with IGF2BP2 shRNA or sh-NC vectors were subcutaneously injected into flank of nude mice.
item1088 .
item1089 .
item1090 .
item1091 Each mouse was anaesthetized with inhaled isoflurane, and the left proximal ureter was exposed. Then, the ureter was ligated with 6-0 silk thread and severed. In the sham operation group, the left ureters of mice were exposed, but not ligated or severed. The 3rd, 7th and 14th days after surgery were the time points for killing. At each time point, a total of 10 mice in the UUO group were executed, and a total of 10 mice in the sham group were also executed to serve as controls.
item1092 NOD/SCID mice (6-week-old) were injected (subcutaneously in both flanks) with 5.0 x 106 PANC-1 GemR cells (infected with scr or METTL14 shRNA) per mouse suspended in 50 ul PBS and mixed with equal volume of growth factor reduced matrigel. One week after injection, we started measuring tumor size at the indicated times. Tumor size was calculated by 0.5 × (long diameter) × (short diameter)2. The mice were treated with vehicle or 100 mg/kg gemcitabine intraperitoneally twice a week.
item1093 The clone, with one wild-type Mettl3 allele and one L1L2_Bact_P cassette inserted allele, was injected into C57BL/6 blastocysts. Mettl3-targeted mouse line was established from a germline-transmitting chimera. The chimeric mouse was crossed to C57BL/6 Flp mice to excise the neomycin resistance system.
item1094 The normal group consisted of C57BL/6J mice on normal diet and the HFD group consisted of ApoE C57BL/6J mice on HFD (containing 10% lard oil, 4% milk powder, 2% cholesterol, and 0.5% sodium cholate). Four weeks after HFD feeding, the mice were injected with 200 ul lentivirus containing 1 × 10-/--/-8 TU (lentivirus carrying sh-METTL3 or sh-NC; designed and constructed by GENCHEM (Shanghai, China)) via tail vein. Six weeks after transfection, all mice were euthanized by a tail vein injection of 200 mg/kg pentobarbital sodium.
item1095 .
item1096 METTL3 knockout or control CT26 and MC38 cells were injected subcutaneously into the dorsal flank of each 4- to 6-week-old male immunocompetent BALB/c and C57BL/6 mice, respectively. Anti-Gr-1 (BE0075; Bio-X-Cell, Lebanon, NH) or immunoglobulin (Ig)G isotype control (BE0090, Bio-XCell) was given every other day via intraperitoneal injection (150 ug/mouse). SB-265610 (Tocris, Bristol, UK) or phosphate-buffered saline was administrated through intraperitoneal injections at a dosage of 2 mg/kg per day. Tumor sizes were measured every other day. To establish an orthotopic mouse model of CRC, 5- to 6-week-old male C57BL/6 mice were treated with 1.7% dextran sodium sulfate in drinking water for 5 days, and then allowed to recover for 3 days. After 24 hours of fasting, METTL3 knockout or control MC38 cells suspended in 50 ul of 1 mg/mL Matrigel-phosphate-buffered saline (Corning, Corning, NY) were instilled into the colon lumen of anesthetized mice, coated sparingly with Vaseline.
item1097 After 50g male NORAD-KO or C57 mice at age of 8 weeks were anesthetized with 3% (w/v) pentobarbital (2 ml/kg) and grouped randomly, investigators blinded to the group allocation performed the experiment. The disc levels in rat tail (Co6/7, 7/8, and 8/9) were located by palpation on the coccygeal vertebrae and confirmed by trial radiography. Needles (33-G) were used to puncture the annulus fibrosus layer though the tail skin, in parallel to the end plates. To ensure that the needle did not penetrate too deeply , the length of the needle was pre-determined according to the dimensions of annulus fibrosus and the NP , which were measured in a preliminary experiment and found to be approximately 4 mm. Five kinds of solution were prepared for intradisc injection, including AA V vector, AAV containing shPUM1, AAV containing shPUM2 for Norad KO mice, AAV vector, AAV containing shE2F3, AAVcontaining OE-E2F3 for WT mice.
item1098 YTHDF3-/- mice were generated using the CRISPR-Cas9 system.
item1099 For the subcutaneous implantation model, 1 × 106 Cal27 cells stably transduced with lentivirus were injected into the left or right flanks of BALB/c nude mice (aged 4-6 weeks). Following stable transfection, 2 × 105 SCC7 cells were subcutaneously inoculated into C3H mice (aged 6-8 weeks).
item1100 .
item1101 Based on our study design, the enrolled animals were blindingly grouped and received the following treatments: (1)Sham-NC vectors, (2)Sham-sh-METTL3, (3)Sham-sh-METTL3 + miR-150, (4)Sham-sh-METTL3 + Lv-YTHDF2, (5)Sham-sh-METTL3 + sh-BDNF, (6)Sham-anti-miR150, (7)Sham-anti-miR150+sh-BDNF, (8)SNI-Lv-METTL3, (9)SNI-Lv-METTL3 + anti-miR-150, (10)SNI-Lv-METTL3 + sh-YTHDF2, (11)SNI-Lv-METTL3 + Lv-BDNF, (12)SNI-miR150, (13)SNI-miR150+Lv-BDNF. The pain behaviors were detected in the respective time points and the expressions of METTL3 and miR-150 were determined at day 14 after the rats were sacrificed.
item1102 After general anesthesia with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (4 mg/kg), topical application of 0.5% proparacaine ophthalmic solution was conducted. Three sutures (10-0 nylon) were placed intrastromally between the limbus and corneal center at the 4, 8, and 12 o'clock positions. Topical norfloxacin was applied after surgery.
item1103 .
item1104 The clone, with one wild-type Mettl3 allele and one L1L2_Bact_P cassette inserted allele, was injected into C57BL/6 blastocysts. Mettl3-targeted mouse line was established from a germline-transmitting chimera. The chimeric mouse was crossed to C57BL/6 Flp mice to excise the neomycin resistance system.
item1105 .
item1106 .
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item1111 .
item1112 .
item1113 .
item1114 .
item1115 Mettl3flox/flox mice were crossed with the transgenic Cdh5-CreERT2 mice to generate the Mettl3-ecKO mice. Cdh5-Cre Mettl3flox/flox mice received an intragastric injection of 50 ul tamoxifen (1 mg/mL) at P1-P3 and P5 for Cre activation and Mettl3 knockout. After Mettl3 knockout, the mouse pups and their nursing mothers were exposed to 75% oxygen (hyperoxia) from P7 to P12 in an incubator chamber. Then, the pups were returned to normal oxygen conditions (normoxia).
item1116 .
item1117 .
item1118 Mice bearing the Alkbh5-floxed allele (Alkbh5 fl/fl , Cyagen) were crossed with transgenic mice expressing Cre recombinase under the control of the Myl1 promoter (Myl1-Cre; Stock No: 024713, The Jackson Laboratory) to generate muscle-specific Alkbh5 knockout mice (Myl1-Cre;Alkbh5 fl/fl ). Littermate Alkbh5fl/fl mice were used as controls. Genotyping by tail DNA and PCR were performed at 4 weeks of age.
item1119 Twenty 5-week-old male nude mice were randomly divided into two groups and injected with either 2B-NNK or 2B-C cells. Tumor growth was measured every 3 days.
item1120 Mice were randomized into several groups. For the subcutaneous implantation model, 1?×?106 cells were injected subcutaneously into the flank regions of female BALB/c nude mice (4-5 weeks). For lung colonization assays, 1 × 106 cells were injected into the tail vein of female NOD/SCID mice (6-7 weeks), and 6 weeks later the lung was removed and fixed with 10% formalin.
item1121 .
item1122 HCC cells stably transfected with empty vector or circCPSF6 were subject to construct animal models, followed by the regular treatment of verteporfin, an inhibitor of YAP signaling.
item1123 For the in vivo cell proliferation assay, A549 and SPCA1 cells were stably transfected with sh-Ctrl and sh-KIAA1429 using lentivirus (GeneChem, Shanghai, China). The cells were subcutaneously injected into either side of the posterior flanks of the mouse. The tumor volume was measured every few days (length×width2×0.5).
item1124 .
item1125 5 × 106 Huh7 and HCC-LM3 cells resuspended in 100ul PBS were subcutaneously injected to the left flank of the mice (randomly selected, five mice per group for Huh7 cells in the first time and ten mice per group for HCC-LM3 cells in the second time. No blinding was performed).
item1126 Cas9 and sgRNA were microinjected into the fertilized eggs of C57BL/6J mice, which were then transplanted to obtain positive F0 mice. The statuses of F0 mice were confirmed by PCR and sequencing. Next, positive F0 mice were mated with C57BL/6J mice to yield stable F1 generation mice. F1 and F2 transgenic mice were used in this study.
item1127 The pump was prefilled with Ang-II or saline and then incubated in sterile saline at 37 ℃ for 48 h. After the mice were anesthetized with 3.0% isoflurane mixed with oxygen, an incision was made on the back skin of the mice, and the pump was implanted into the subcutaneous area, followed by suturing the incision. After the operation, the mice were given buprenorphine (0.1 mg/kg) to reach analgesia. Finally, the regaining consciousness mice were returned to cages and fed until the end of the experiment.
item1128 The pump was prefilled with Ang-II or saline and then incubated in sterile saline at 37 ℃ for 48 h. After the mice were anesthetized with 3.0% isoflurane mixed with oxygen, an incision was made on the back skin of the mice, and the pump was implanted into the subcutaneous area, followed by suturing the incision. After the operation, the mice were given buprenorphine (0.1 mg/kg) to reach analgesia. Finally, the regaining consciousness mice were returned to cages and fed until the end of the experiment.
item1129 For the subcutaneous implantation ICC mouse model, 6-week-old male NCG mice (Jiangsu, China) were randomly enrolled into shNC group and shYTHDF1 group (n = 9); 1 × 106 HuCCT1 cells in 0.1-mL PBS transfected with shNC or shYTHDF1 were subcutaneously inoculated in the right flanks of the mice. For AKT/YapS127A-induced orthotopic ICC mouse model, 16 mice were divided into two groups randomly. For the control group, 20-ug AKT, 30-ug Yap, and 2-ug pCMV/SB plasmids plus 20-ug vector plasmids as control were diluted in 2-mL saline and then were injected into the lateral tail vein within 7 s. For the YTHDF1-overexpressed group, mice were injected with additional 20-ug YTHDF1 plasmids under the same conditions. Mice were sacrificed at 4 weeks after injection, and liver tissues were harvested for analysis.
item1130 .
item1131 .
item1132 .
item1133 .
item1134 FTO over-expression db/db mice were generated through injecting the tail vein with Fto-overexpression lentivirus at 12 weeks.
item1135 .
item1136 The PC cell line PANC1 was subcutaneously injected into the dorsal flank of the mice at the concentration of 1 × 106 cells per mouse.
item1137 .
item1138 The PC cell line PANC1 was subcutaneously injected into the dorsal flank of the mice at the concentration of 1 × 106 cells per mouse.
item1139 .
item1140 First, subcutaneous transplanted model was used to evaluate the growth of BT-549LMF3 and BT-549 cells. Cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 ul PBS + 200 ul Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient female mice. Second, subcutaneous transplanted model was used to evaluate the metastasis potential of BT-549LMF3 and BT-549 cells. Cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 ul PBS + 200 ul Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient female mice. Third, the in vivo lung metastasis model was established by injecting with BT-549, BT-549LMF3, FTO stable BT-549LMF3, sh-METTL3 BT-549LMF3, and sh-KRT7 BT-549LMF3 stable cells (1 × 106 per mouse, n = 5 for each group)
item1141 First, subcutaneous transplanted model was used to evaluate the growth of BT-549LMF3 and BT-549 cells. Cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 ul PBS + 200 ul Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient female mice. Second, subcutaneous transplanted model was used to evaluate the metastasis potential of BT-549LMF3 and BT-549 cells. Cells (5 × 106 per mouse, n = 5 for each group) were diluted in 200 ul PBS + 200 ul Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient female mice. Third, the in vivo lung metastasis model was established by injecting with BT-549, BT-549LMF3, FTO stable BT-549LMF3, sh-METTL3 BT-549LMF3, and sh-KRT7 BT-549LMF3 stable cells (1 × 106 per mouse, n = 5 for each group)
item1142 Xenograft tumor formation was observed in 5 of 5 and 3 of 5 animals when 1 × 105 and 1 × 104 sorted LGR5+ cells, were subcutaneously injected into nude mice, respectively
item1143 .
item1144 Mice were injected subcutaneously with LoVo (1 × 106) cells, which were stably transfected with the control shRNA or YTHDF1 shRNA.
item1145 .
item1146 .
item1147 For tumour xenograft models, 1 × 107 HuCC-T1 cells in knockdown group or control group were implanted into the right flank of 5-week-old female nude mice. The volumes of tumour were recorded every 4 days by calliper. The volumes were calculated as length × width2/2. For patient-derived xenograft (PDX) model (PDX0075), ICC tissues from a patient, who relapsed in 6 months after R0 resection and subsequent chemotherapy with cisplatin and gemcitabine, were diced into 3 mm3 pieces and transplanted subcutaneously into the right flank of 5-week-old female B-NDG mice.
item1148 .
item1149 For tumor growth assay, 4 × 106 logarithmically growing GIST cells were transfected with T1S-vector, T1S-METTL3, 882S-vector, or 882S-METTL3 constructs, and subcutaneously injected in 100 ul of PBS into the flank of female nude mice(4-week-old). Mice were then randomly divided into 8 groups (n = 5 in each group): (1) injected with T1S-vector-harboring cells, and treated with imatinib (600 mg/l in drinking water); (2) injected with T1S-vector-harboring cells, and treated with imatinib (600 mg/l in drinking water) and MRP1 inhibitor (100 mg/l in drinking water); (3) injected with T1S-METTL3-harboring cells, and treated with imatinib (600 mg/l in drinking water); (4) injected with T1S-METTL3-harboring cells, and treated with imatinib (600 mg/l in drinking water) and MRP1 inhibitor (100 mg/l in drinking water); (5) injected with 882S-vector-harboring cells and treated with imatinib (600 mg/l in drinking water); (6) injected with 882S-vector-harboring cells, and treated with imatinib (600 mg/l in drinking water) and MRP1 inhibitor (100 mg/l in drinking water); (7) injected with 882S-METTL3-harboring cells, and treated with imatinib (600 mg/l in drinking water); and (8) injected with 882S-METTL3-harboring cells, and treated with imatinib (600 mg/l in drinking water) and MRP1 inhibitor (100 mg/l in drinking water).
item1150 For subcutaneous transplanted model, sh-control and sh-METTL3 HCT-116/5-FU cells (5 × 106 per mouse) were diluted in 100ul PBS + 100 ul Matrigel (BD Biosciences, San Jose, CA, USA) and injected subcutaneously in the rear flank fat pad of the nude mice.
item1151 A total of 5 × 106 cells in 200 ul PBS were injected subcutaneously into the flanks of nude mice. After injection, cisplatin treatment was initiated on day 5. Mice were injected with 5 mg/kg cisplatin or PBS solution in the abdominal cavity once a week for 3?weeks.
item1152 .
item1153 Using the diabetic rat model established by HGHF feeding with a subsequent intraperitoneal injection of a single low dose of streptozocin.
item1154 .
item1155 .
item1156 .
item1157 .
item1158 Mettl3flox/flox and Mettl3-HKO mice were fasted overnight and then injected intraperitoneally with 20 uM BODIPY FL C16 in 200 ul saline for 20 min.
item1159 .
item1160 Mettl3flox/flox and Mettl3-HKO mice were fasted overnight and then injected intraperitoneally with 20 uM BODIPY FL C16 in 200 ul saline for 20 min.
item1161 .
item1162 For the tumor xenograft, mice were randomly divided into three groups with four mice for each group. Then, 1 × 106 cells after indicated treatment were harvested and resuspended in 50 ul of PBS. Then the cells were subcutaneously injected into the right front flank of each mouse.
item1163 .
item1164 For CDX model, nude mice (female, 4-6-week-old) were subcutaneously injected with 5 × 106 HCT116 cells on the both flank. For PDX model, the patient tumors were divided into small pieces and then inoculated on both flank of nude mice. For knockdown FTO mice model, FTO mice model, two weeks after inoculation, the shFTO#3 lenti-virus injected into the tumor for three consecutive days. For combined medication mice model, intraperitoneal injection of Rhein and Olaparib was started one week after inoculation
item1165 For CDX model, nude mice (female, 4-6-week-old) were subcutaneously injected with 5 × 106 HCT116 cells on the both flank. For PDX model, the patient tumors were divided into small pieces and then inoculated on both flank of nude mice. For knockdown FTO mice model, FTO mice model, two weeks after inoculation, the shFTO#3 lenti-virus injected into the tumor for three consecutive days. For combined medication mice model, intraperitoneal injection of Rhein and Olaparib was started one week after inoculation
item1166 .
item1167 5 × 106 SW1990 cells expressing NUCB1 (oeNUCB1) or control vector (oeNC) were injected subcutaneously.
item1168 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1169 .
item1170 Male nu/nu mice between 4 and 6 weeks of age received subcutaneous injections of equivalent AGS cells expressing either control or LV-HOXB13 within 30 min of harvesting on the right and left flanks.
item1171 Twenty-four specific pathogen free female BALB/c nude mice (age: 6 weeks, weight: 15 ~ 18 g) were purchased from Slac Laboratory Animal Co., Ltd., and subcutaneously injected with SW620 cells stably transfected with oe-NC, oe-GSK3-Beta + oe-NC, or oe-GSK3-Beta + oe-c-Myc to establish a subcutaneous xenograft tumour model in nude mice.
item1172 Twenty-four specific pathogen free female BALB/c nude mice (age: 6 weeks, weight: 15 ~ 18 g) were purchased from Slac Laboratory Animal Co., Ltd., and subcutaneously injected with SW620 cells stably transfected with oe-NC, oe-GSK3-Beta + oe-NC, or oe-GSK3-Beta + oe-c-Myc to establish a subcutaneous xenograft tumour model in nude mice.
item1173 .
item1174 .
item1175 Approximately 5 × 106 253J and 5637 cells infected with indicated vectors were injected subcutaneously into the flank of the mice.
item1176 Homologous recombination was used, cas9 mRNA, gRNA, and donor vector were microinjected into the fertilized eggs of C57BL/6J mice to obtain F0 generation mice.
item1177 .
item1178 Each mouse was injected subcutaneously with 5 × 106 units of tumor cells to construct the tumor model.
item1179 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1180 To detect the effect of NKILA on CAA growth, 5 × 106 control and NKILA-depleted HuCCT1 cells (n = 4 per group) were subcutaneously injected into male BALB/c nude mice (4-6 weeks old).
item1181 .
item1182 .
item1183 circ_0008542, where rats were injected with exosomes containing circ_0008542 into the tail vein for 8 weeks.
item1184 circ_0008542, where rats were injected with exosomes containing circ_0008542 into the tail vein for 8 weeks.
item1185 .
item1186 At 1 week post-injection with PC-3 cells, mice were randomly assigned to three groups (n = 8 per group): the ASO-NC group (injection with ASO negative control targeting unknown sequence, 5 nmol in 100 uL PBS for each mouse), the ASO-L group (injection with low-dose ASO targeting PCAT6, 5 nmol in 100 uL PBS for each mouse), and the ASO-H group (injection with high-dose ASO targeting PCAT6, 10 nmol in 100 uL PBS for each mouse).
item1187 At 1 week post-injection with PC-3 cells, mice were randomly assigned to three groups (n = 8 per group): the ASO-NC group (injection with ASO negative control targeting unknown sequence, 5 nmol in 100 uL PBS for each mouse), the ASO-L group (injection with low-dose ASO targeting PCAT6, 5 nmol in 100 uL PBS for each mouse), and the ASO-H group (injection with high-dose ASO targeting PCAT6, 10 nmol in 100 uL PBS for each mouse).
item1188 At 1 week post-injection with PC-3 cells, mice were randomly assigned to three groups (n = 8 per group): the ASO-NC group (injection with ASO negative control targeting unknown sequence, 5 nmol in 100 uL PBS for each mouse), the ASO-L group (injection with low-dose ASO targeting PCAT6, 5 nmol in 100 uL PBS for each mouse), and the ASO-H group (injection with high-dose ASO targeting PCAT6, 10 nmol in 100 uL PBS for each mouse).
item1189 At 1 week post-injection with PC-3 cells, mice were randomly assigned to three groups (n = 8 per group): the ASO-NC group (injection with ASO negative control targeting unknown sequence, 5 nmol in 100 uL PBS for each mouse), the ASO-L group (injection with low-dose ASO targeting PCAT6, 5 nmol in 100 uL PBS for each mouse), and the ASO-H group (injection with high-dose ASO targeting PCAT6, 10 nmol in 100 uL PBS for each mouse).
item1190 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1191 .
item1192 .
item1193 The mice were subcutaneously inoculated with 786-O cells stably transfected with lentiviruses carrying sh-NC/sh-circMET, respectively (5 × 106, 200 uL).
item1194 .
item1195 .
item1196 For tumor xenograft studies, 143B cells stably expressing scrambled shRNA or ALKBH5 shRNA (1 × 106) were injected subcutaneously into the flanks of 4-week-old athymic nude mice.
item1197 For tumor xenograft studies, 143B cells stably expressing scrambled shRNA or ALKBH5 shRNA (1 × 106) were injected subcutaneously into the flanks of 4-week-old athymic nude mice.
item1198 Twenty mice were randomly divided into three groups: normal mice group (N), diabetic mice group (DM), and diabetic mice administrated with TSA group (DM + TSA).
item1199 .
item1200 For the studies of investigating mice survival, mice were intracranially injected with 10,000 GSC11, 10,000 GSC7-2, or 500,000 LN229 cells.
item1201 5 × 106 infected T98G cells (LV-NC or LV-YTHDF2) were injected into the flanks of mice through subcutaneous.
item1202 For the studies of investigating mice survival, mice were intracranially injected with 10,000 GSC11, 10,000 GSC7-2, or 500,000 LN229 cells.
item1203 Fresh PDX tumor samples collected from two established PDX models (PDX #07 with high TRIB2 expression and PDX #12 with low TRIB2, passages three to four) were minced and subcutaneously implanted into the flanks of 3- to 4-week-old female BALB/c nude mice (Jiesijie Laboratory Animals).
item1204 Fresh PDX tumor samples collected from two established PDX models (PDX #07 with high TRIB2 expression and PDX #12 with low TRIB2, passages three to four) were minced and subcutaneously implanted into the flanks of 3- to 4-week-old female BALB/c nude mice (Jiesijie Laboratory Animals).
item1205 .
item1206 BALB/c nude mice which were co-injected with THP-1 cells and PATU-8988 cells subcutaneously.
item1207 BALB/c nude mice which were co-injected with THP-1 cells and PATU-8988 cells subcutaneously.
item1208 BALB/c nude mice which were co-injected with THP-1 cells and PATU-8988 cells subcutaneously.
item1209 For lung metastasis model, 1 × 106 HCC cells suspended in 100 ul serum free DMEM were injected via the tail vein of nude mice.
item1210 For lung metastasis model, 1 × 106 HCC cells suspended in 100 ul serum free DMEM were injected via the tail vein of nude mice.
item1211 .
item1212 5 × 106 infected T98G cells (LV-NC or LV-YTHDF2) were injected into the flanks of mice through subcutaneous.
item1213 .
item1214 .
item1215 A total of 5 × 106 786O cells were subcutaneously injected into the left flanks of the mice.
item1216 Mice with a Tmem30a deletion specifically in pancreatic beta cells were generated as previously described. Mice developed with NAFLD were named for Tmem30a-associated NAFLD (TAN) mice. The littermate mice with genotypes of Tmem30aloxP/loxP were used as controls.
item1217 Mice with a Tmem30a deletion specifically in pancreatic beta cells were generated as previously described. Mice developed with NAFLD were named for Tmem30a-associated NAFLD (TAN) mice. The littermate mice with genotypes of Tmem30aloxP/loxP were used as controls.
item1218 Mice with a Tmem30a deletion specifically in pancreatic beta cells were generated as previously described. Mice developed with NAFLD were named for Tmem30a-associated NAFLD (TAN) mice. The littermate mice with genotypes of Tmem30aloxP/loxP were used as controls.
item1219 Mice with a Tmem30a deletion specifically in pancreatic beta cells were generated as previously described. Mice developed with NAFLD were named for Tmem30a-associated NAFLD (TAN) mice. The littermate mice with genotypes of Tmem30aloxP/loxP were used as controls.
item1220 5 × 105 cells were injected subcutaneously into the right axilla of mice. Tumor volume was measured by a caliper weekly and calculated as length × width2 × 0.52. For the liver orthotopic-implanted models, each liver of mice was injected with 1 × 106 cells.
item1221 1 × 105 HNE2 cells (with or without METTL3 knockdown) were labeled with luciferase gene and injected into the tail vein of the nude mice.
item1222 1 × 105 HNE2 cells (with or without METTL3 knockdown) were labeled with luciferase gene and injected into the tail vein of the nude mice.
item1223 For the xenograft tumor model, approximately 1 × 106 ccRCC cells suspended in 100 uL PBS were subcutaneously inoculated in the right flank of 5-week-old BALB/c nude mice. For the ccRCC orthotopic implantation model, approximately 1 × 106 ccRCC cells suspended in 30 uL Matrigel were injected under the renal capsule of 5-week-old BALB/c nude mice. After 6 weeks, the anesthetized mice were intraperitoneally injected with D-luciferin (Yeason) and imaged using an in vivo imaging system to detect tumor growth and metastasis. For the lung metastasis model, approximately 5 × 105 ccRCC cells suspended in PBS were injected into the tail vein of 5-week-old mice. After 6-8 weeks, mice were anesthetized and lung metastasis was imaged as above.
item1224 1 × 105 HNE2 cells (with or without METTL3 knockdown) were labeled with luciferase gene and injected into the tail vein of the nude mice.
item1225 1 × 105 HNE2 cells (with or without METTL3 knockdown) were labeled with luciferase gene and injected into the tail vein of the nude mice.
item1226 A total of 1 × 106 luciferase-labeled BC cells transfected with shWTAP or shNC were injected subcutaneously with or without C5aR1 neutrophils (tumor cells:neutrophils, 10:1).
item1227 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1228 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1229 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1230 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1231 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1232 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1233 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1234 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1235 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1236 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1237 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1238 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1239 2 × 106 CT26 cells with knockout of Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1, or Mettl14/Irf1 and control were suspended in 200 uL of PBS/Matrigel (Corning) (1:1) and then subcutaneously inoculated into flank of each mouse.
item1240 Equal amount of HCT116 cells (2 × 106) stably expression of relevant plasmids was injected into the right flank of mice, tumor bulks was monitored once a week after injection and volumes were counted as 0.5 × a2 × b (a and b respectively indicated short and long diameter of tumor).
item1241 Equal amount of HCT116 cells (2 × 106) stably expression of relevant plasmids was injected into the right flank of mice, tumor bulks was monitored once a week after injection and volumes were counted as 0.5 × a2 × b (a and b respectively indicated short and long diameter of tumor).
item1242 A total of 106 LLC cells transfected with the following constructs were injected subcutaneously into C57BL/6 mice.
item1243 A total of 106 LLC cells transfected with the following constructs were injected subcutaneously into C57BL/6 mice.
item1244 .
item1245 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1246 .
item1247 To establish the adjuvant-induced arthritis (AIA) model, the rats were given complete Freund's adjuvant (CFA; Chondrex, Inc.) on the left paw of 0.1 ml per 100 g of body weight. Additionally, the rats were injected with normal saline to create the negative control (NC) group.
item1248 To establish the adjuvant-induced arthritis (AIA) model, the rats were given complete Freund's adjuvant (CFA; Chondrex, Inc.) on the left paw of 0.1 ml per 100 g of body weight. Additionally, the rats were injected with normal saline to create the negative control (NC) group.
item1249 To establish the adjuvant-induced arthritis (AIA) model, the rats were given complete Freund's adjuvant (CFA; Chondrex, Inc.) on the left paw of 0.1 ml per 100 g of body weight. Additionally, the rats were injected with normal saline to create the negative control (NC) group.
item1250 .
item1251 .
item1252 .
item1253 .
item1254 .
item1255 2 × 106 stably transfected HeLa cells were subcutaneously inoculated into the left flank of mice.
item1256 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1257 2 × 106 stably transfected HeLa cells were subcutaneously inoculated into the left flank of mice.
item1258 The cells (1 × 106) were re-suspended in normal saline and mixed with 25% Matrigel matrix (50 uL) at a 1:1 ratio and subcutaneously injected into the right groin of the mice.
item1259 Stably transfected shMETTL14 and shNC DU145 cells (5×106 cells) suspended in a mixture of 100uL PBS were subcutaneously injected into the right flank of male nude BALB/C mice (6-8 weeks old) to induce tumor formation.
item1260 Stably transfected shMETTL14 and shNC DU145 cells (5×106 cells) suspended in a mixture of 100uL PBS were subcutaneously injected into the right flank of male nude BALB/C mice (6-8 weeks old) to induce tumor formation.
item1261 The vitreous cavity of the right eye of each rat was injected either with ARPE-19 cells (1 × 105 per uL in PBS [pH 7.4]) overexpressing METTL3 or control vector cells or with an equal volume of PBS (pH 7.4).
item1262 .
item1263 .
item1264 .
item1265 T24 cells were subcutaneously injected into the mice (1 x 106 cells / injecting site).
item1266 2 × 106 U87MG cells already expressing shscr or shADAR1 were subcutaneously injected in the flank of 6-week-old nude mice (nu/nu, Charles River, Wilmington, MA, USA).
item1267 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1268 Male mice (8-10 weeks old, SLAC Laboratory Animal Co., Ltd., Shanghai, China) were administrated with STZ through intraperitoneal injection (I.P) for continuous 5 days (d).
item1269 .
item1270 .
item1271 .
item1272 .
item1273 To generate a highly metastatic orthotopic xenograft model, 1 × 105 luciferase-expressing Renca cells (Luc-Renca) in 25 ul of 2:1 (v/v) PBS:Matrigel were injected into the right sub-renal capsule of the kidney of BALB/c mice (4 mice/group).
item1274 FTO-overexpressing and control cells (2 × 106 suspended in 100 ul PBS) were subcutaneously injected into each mouse.
item1275 FTO-overexpressing and control cells (2 × 106 suspended in 100 ul PBS) were subcutaneously injected into each mouse.
item1276 Female BALB/c nude mice aged 5 weeks (Beijing Vital River Laboratory Animal Technology) were randomly grouped and injected subcutaneously with 0.1 mL of cell suspension containing 2 × 106 PDAC cells in the back flank.
item1277 The mice were randomly divided into two groups which were inoculated with stable YTHDF2-expressing LUSC cells and the vector LUSC cells. A total of 5 × 106 of the cells were suspended in 0.1 ml of PBS and then injected subcutaneously into the flanks of mice.
item1278 5 × 106 SW1990 cells expressing NUCB1 (oeNUCB1) or control vector (oeNC) were injected subcutaneously.
item1279 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1280 All the mice were randomly divided into three groups: the normal saline group (NS), the lipopolysaccharide + negative control group (LPS + NC), and the LPS + miR-29a-3p agomir group (LPS + Agomir).
item1281 The SKG mice were randomly divided into three groups: a PBS group, an Av-NC group, and an Av-ELMO1 group. The SKG mice in the Av-ELMO1 group were treated with 5 × 1010 Av-ELMO1 via intravenous tail vein injection at the time of disease induction, and the SKG mice in the Av-NC group or PBS group were separately treated with equal amounts of control adenoviruses or PBS.
item1282 .
item1283 .
item1284 For the formation of xenograft tumors, 5 × 106 AGS cells mixed in Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were subcutaneously injected into BALB/c nude mice (5-week-old male).
item1285 .
item1286 For melittin treatment study, 4-week-old female BALB/c nude mice were subcutaneously injected with 1 × 107 T24 or BIU87 cells.
item1287 For the in vivo metastasis assays, luciferase labeled OS-RC-2 cells stably expressing OE-P2RX6 or pWPI-vector were injected into the tail vein of 5 weeks old BALB/c nude mice (Sipper-BK laboratory animal Company, Shanghai, China).
item1288 To generate the Mettl3+/- mice, 2 specific single-guide RNAs (sgRNAs) targeting exon 2 of Mettl3 were used, which resulted in a frameshift mutation and generated a premature stop codon.
item1289 MG63 cells transduced with lentivirus expressing shTRIM7 or shNC, and SAOS2 cells transduced with lentivirus expressing TRIM7, BRMS1, TRIM7 plus BRMS1 or control vector, were injected via the tail vein into the nude mice (1 × 106 cells/mouse) (n = 11 per group).
item1290 100,000 pLKO and PARP1-sh1 (PT1 and PT2) cells were mixed with matrix gel and inoculate into BALB/C nude mice, respectively. After 25 days, 6 organoid transplanted tumor mice were treated with oxaliplatin (Sellekchem, s1224) twice a week for 4 weeks at a dose of 5 mg/kg.
item1291 MG63 cells transduced with lentivirus expressing shTRIM7 or shNC, and SAOS2 cells transduced with lentivirus expressing TRIM7, BRMS1, TRIM7 plus BRMS1 or control vector, were injected via the tail vein into the nude mice (1 × 106 cells/mouse) (n = 11 per group).
item1292 MG63 cells transduced with lentivirus expressing shTRIM7 or shNC, and SAOS2 cells transduced with lentivirus expressing TRIM7, BRMS1, TRIM7 plus BRMS1 or control vector, were injected via the tail vein into the nude mice (1 × 106 cells/mouse) (n = 11 per group).
item1293 For subcutaneous xenograft models, 0.1 mL of cell suspension containing 106 cells were injected subcutaneously into the right flank of mice (n = 6 for each group).
item1294 Male BALB/c mice were grouped into control, CS, CS+AAV-ShMETTL3, and CS+AAV-NC shRNA groups, n = 6 animals per group. Mice in the AAV-ShMETTL3 and AAV-NC shRNA (GeneChem, China) groups were dosed by intranasal instillation after CS exposure for 4 weeks.
item1295 Twenty-four female BALB/c athymic nude mice, which were 4-6 weeks old and weighed 20.0-25.0 g, were obtained from the Animal Research Center of PUMCH. WTAP-OE, WTAP-NC, shWTAP and shNC-lentivirus infected MIA PaCa-2 cells (5 × 106) were suspended in 50 uL PBS and then injected subcapsularly into the pancreatic tissue by 1-mL syringes.
item1296 As to the subcutaneous transplanted model, WT Vec, Mettl3 Mut/-+vec, WTPDK4, Mettl3 Mut/-+vecPDK4 cells (2 × 10 6 per mouse, n = 10 for each group) were diluted in 200 uL normal medium + 200 uL Matrigel (BD Biosciences) and subcutaneously injected into immunodeficient mice to investigate tumor growth.
item1297 1 × 107 SK-HEP-1-Luc-shControl or SK-HEP-1-Luc-shMETTL3 stable cells were suspended in 300 uL of PBS and injected orthotopically into the left liver lobe of nude mice.
item1298 The caudal vein injection mouse model, intrasplenic injection mouse model, and orthotopic xenograft IRFA HCC mouse models, including patient-derived xenograft (PDX), and cell-line-derived xenograft implantation models, were established as reported.
item1299 5 × 106 cells were suspended in 100 uL PBS and then were inoculated subcutaneously.
item1300 .
item1301 100,000 pLKO and PARP1-sh1 (PT1 and PT2) cells were mixed with matrix gel and inoculate into BALB/C nude mice, respectively. After 25 days, 6 organoid transplanted tumor mice were treated with oxaliplatin (Sellekchem, s1224) twice a week for 4 weeks at a dose of 5 mg/kg.
item1302 .
item1303 .
item1304 .
item1305 .
item1306 .
item1307 .
item1308 .
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item1312 1×106 MDA-MB-231 cells were resuspended in 100 uL PBS with 50% Matrigel (Corning Costar, USA), and injected into the mammary fat pad of the mice.
item1313 Modelled two groups of female 6-week-old BALB/C mice: severe asthma group and blank control group (n = 3 per group). They had the same feeding conditions and growth environment. Immunization solution: Dissolve 20 mg ovalbumin (OVA) in 1 ml normal saline (NS), after OVA is completely dissolved, dilute 0.4-10 ml and mix well, then it was mixed with the same volume of liquid aluminium adjuvant and placed on a shaking table at 4℃ for 30 min. Challenge solution: Add 0.5 g OVA into 10 ml NS, fully dissolve it, and shake it on a shaking table at 4℃ for 30 min. Immunization: Mice were injected intraperitoneally on days 0 and 12, each with 0.2 ml; the control group was treated with equal volume of normal saline. Challenge: On days 18-23, the mice were atomized by ultrasound in a closed container at a dose of 10 ml once a day for 20 min. Lung tissue was taken 24 h after the last atomization and immediately stored in liquid nitrogen.
item1314 Modelled two groups of female 6-week-old BALB/C mice: severe asthma group and blank control group (n = 3 per group). They had the same feeding conditions and growth environment. Immunization solution: Dissolve 20 mg ovalbumin (OVA) in 1 ml normal saline (NS), after OVA is completely dissolved, dilute 0.4-10 ml and mix well, then it was mixed with the same volume of liquid aluminium adjuvant and placed on a shaking table at 4℃ for 30 min. Challenge solution: Add 0.5 g OVA into 10 ml NS, fully dissolve it, and shake it on a shaking table at 4℃ for 30 min. Immunization: Mice were injected intraperitoneally on days 0 and 12, each with 0.2 ml; the control group was treated with equal volume of normal saline. Challenge: On days 18-23, the mice were atomized by ultrasound in a closed container at a dose of 10 ml once a day for 20 min. Lung tissue was taken 24 h after the last atomization and immediately stored in liquid nitrogen.
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item1321 Animals were bred and housed in the pathogen-free facility of the Laboratory Animal Center of Shanghai General Hospital (Shanghai, China). All lungs were collected 4 weeks after BLM treatment for histology and further study. Lung microsections (5 uM) were applied to Masson's trichrome and Sirius red staining to visualize fibrotic lesions.
item1322 Animals were bred and housed in the pathogen-free facility of the Laboratory Animal Center of Shanghai General Hospital (Shanghai, China). All lungs were collected 4 weeks after BLM treatment for histology and further study. Lung microsections (5 uM) were applied to Masson's trichrome and Sirius red staining to visualize fibrotic lesions.
item1323 1×106 MDA-MB-231 cells were resuspended in 100 uL PBS with 50% Matrigel (Corning Costar, USA), and injected into the mammary fat pad of the mice.
item1324 At 8 weeks of age, METTL14 cKO and WT mice were challenged with LPS (Sigma-Aldrich, St. Louis, MO; L2880, single intraperitoneal injection at 5 mg/kg, n = 3) or CCl4 (10%, Macklin, Shanghai, China; C805332, intraperitoneal injection at 5 mL/kg diluted with corn oil, twice per week for 4 weeks, n = 3). The corresponding control groups were treated with single intraperitoneal injection of saline (n = 3) or intraperitoneal injection of corn oil twice per week for 4 weeks (n = 3), respectively. Two hours after LPS injection and 4 weeks after CCl4 treatment, METTL14 cKO and WT mice were etherized and the primary KCs were isolated from liver according to a previously published method.
item1325 At 8 weeks of age, METTL14 cKO and WT mice were challenged with LPS (Sigma-Aldrich, St. Louis, MO; L2880, single intraperitoneal injection at 5 mg/kg, n = 3) or CCl4 (10%, Macklin, Shanghai, China; C805332, intraperitoneal injection at 5 mL/kg diluted with corn oil, twice per week for 4 weeks, n = 3). The corresponding control groups were treated with single intraperitoneal injection of saline (n = 3) or intraperitoneal injection of corn oil twice per week for 4 weeks (n = 3), respectively. Two hours after LPS injection and 4 weeks after CCl4 treatment, METTL14 cKO and WT mice were etherized and the primary KCs were isolated from liver according to a previously published method.
item1326 Liver-specific Bmal1f/f-AlbCre-knockout mice were purchased from Jackson Laboratory. C57BI/6J or Bmal1f/f-AlbCre-knockout male mice were maintained under a 12 hr light/12 hr dark (LD) cycle (ZT0 = 6 AM) and fed ad libitum with normal rodent chow (2018 Global 18% Protein diet, Envigo) and water. At 10-14 weeks of age, 10 male mice per group were sacrificed via CO2 asphyxiation at Zeitgeber Time (ZT) 0,2,6,10,12,14,18,22. In order to induce high levels of ROS in the liver, WT male mice were fasted 12 h and followed by intraperitoneal injection with 300 mg/kg APAP dissolved in PBS and re-fed.
item1327 Liver-specific Bmal1f/f-AlbCre-knockout mice were purchased from Jackson Laboratory. C57BI/6J or Bmal1f/f-AlbCre-knockout male mice were maintained under a 12 hr light/12 hr dark (LD) cycle (ZT0 = 6 AM) and fed ad libitum with normal rodent chow (2018 Global 18% Protein diet, Envigo) and water. At 10-14 weeks of age, 10 male mice per group were sacrificed via CO2 asphyxiation at Zeitgeber Time (ZT) 0,2,6,10,12,14,18,22. In order to induce high levels of ROS in the liver, WT male mice were fasted 12 h and followed by intraperitoneal injection with 300 mg/kg APAP dissolved in PBS and re-fed.
item1328 Liver-specific Bmal1f/f-AlbCre-knockout mice were purchased from Jackson Laboratory. C57BI/6J or Bmal1f/f-AlbCre-knockout male mice were maintained under a 12 hr light/12 hr dark (LD) cycle (ZT0 = 6 AM) and fed ad libitum with normal rodent chow (2018 Global 18% Protein diet, Envigo) and water. At 10-14 weeks of age, 10 male mice per group were sacrificed via CO2 asphyxiation at Zeitgeber Time (ZT) 0,2,6,10,12,14,18,22. In order to induce high levels of ROS in the liver, WT male mice were fasted 12 h and followed by intraperitoneal injection with 300 mg/kg APAP dissolved in PBS and re-fed.
item1329 Liver-specific Bmal1f/f-AlbCre-knockout mice were purchased from Jackson Laboratory. C57BI/6J or Bmal1f/f-AlbCre-knockout male mice were maintained under a 12 hr light/12 hr dark (LD) cycle (ZT0 = 6 AM) and fed ad libitum with normal rodent chow (2018 Global 18% Protein diet, Envigo) and water. At 10-14 weeks of age, 10 male mice per group were sacrificed via CO2 asphyxiation at Zeitgeber Time (ZT) 0,2,6,10,12,14,18,22. In order to induce high levels of ROS in the liver, WT male mice were fasted 12 h and followed by intraperitoneal injection with 300 mg/kg APAP dissolved in PBS and re-fed.
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item1334 For melittin treatment study, 4-week-old female BALB/c nude mice were subcutaneously injected with 1 × 107 T24 or BIU87 cells.
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item1344 For melittin treatment study, 4-week-old female BALB/c nude mice were subcutaneously injected with 1 × 107 T24 or BIU87 cells.
item1345 Leptin receptor-deficient (db/db) mice and control mice (db/ +) were purchased from Shanghai Model Organisms Center, which genetic background is C57BL/6J. All mice were used for experiments at 8-12 weeks old and were housed in constant 24 degrees cages with a 12 h alternating light/dark cycle and free access to water and food. To construct a diabetic heart disease model (DCM), mice were continuously fed to 24 weeks of age, then euthanized, hearts collected in 1.5 ml RNase-free centrifuge tubes, immediately immersed in liquid nitrogen to prevent RNA degradation, and finally stored at 80℃. Five pairs of db/db and db/ + heart samples were selected for RNA sequencing, and the remaining samples were saved for validation.
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item1350 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of 6-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck Millipore, 217503), 0.9% normal saline (Saline) were used as control. The regenerated muscles were collected at day 1, 3, 5, and 10 post-injection. TA muscles were isolated for Hematoxylin and eosin staining or frozen in liquid nitrogen for RNA and protein extraction.
item1351 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of 6-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck Millipore, 217503), 0.9% normal saline (Saline) were used as control. The regenerated muscles were collected at day 1, 3, 5, and 10 post-injection. TA muscles were isolated for Hematoxylin and eosin staining or frozen in liquid nitrogen for RNA and protein extraction.
item1352 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of 6-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck Millipore, 217503), 0.9% normal saline (Saline) were used as control. The regenerated muscles were collected at day 1, 3, 5, and 10 post-injection. TA muscles were isolated for Hematoxylin and eosin staining or frozen in liquid nitrogen for RNA and protein extraction.
item1353 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of 6-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck Millipore, 217503), 0.9% normal saline (Saline) were used as control. The regenerated muscles were collected at day 1, 3, 5, and 10 post-injection. TA muscles were isolated for Hematoxylin and eosin staining or frozen in liquid nitrogen for RNA and protein extraction.
item1354 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of 6-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck Millipore, 217503), 0.9% normal saline (Saline) were used as control. The regenerated muscles were collected at day 1, 3, 5, and 10 post-injection. TA muscles were isolated for Hematoxylin and eosin staining or frozen in liquid nitrogen for RNA and protein extraction.
item1355 For melittin treatment study, 4-week-old female BALB/c nude mice were subcutaneously injected with 1 × 107 T24 or BIU87 cells.
item1356 For mouse muscle injury and regeneration experiment, tibialis anterior (TA) muscles of 6-week-old male mice were injected with 25 uL of 10 uM cardiotoxin (CTX, Merck Millipore, 217503), 0.9% normal saline (Saline) were used as control. The regenerated muscles were collected at day 1, 3, 5, and 10 post-injection. TA muscles were isolated for Hematoxylin and eosin staining or frozen in liquid nitrogen for RNA and protein extraction.
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item1364 Nude mice (4-6 week-old) were administered sterile water and feed in a specific pathogen-free barrier. Using a 1-mL syringe, 1 × 107 HEPG2 cells were subcutaneously inoculated into the right axilla of nude mice to build the HCC xenograft model. When the tumor volume reached 50 mm3, the nude mice were randomly divided into 1 control (n = 4) and 3 treatment groups (n = 4 each). RG7112, apatinib, and RG7112 + apatinib were administered to the treatment groups and an equal volume of dimethyl sulfoxide to the control group by daily gavage for 14 d. The tumor length (L) and width (W) were measured on alternate days using vernier calipers. The following formula was used to calculate the tumor volume: volume (mm3) = 0.5 × L × W × W. At the end of the experiment, the nude mice were killed by CO2 overdose anesthesia. The tumors were dissected and weighed using a precision balance, and the tumor tissue was stored in liquid nitrogen for further analysis.
item1365 Nude mice (4-6 week-old) were administered sterile water and feed in a specific pathogen-free barrier. Using a 1-mL syringe, 1 × 107 HEPG2 cells were subcutaneously inoculated into the right axilla of nude mice to build the HCC xenograft model. When the tumor volume reached 50 mm3, the nude mice were randomly divided into 1 control (n = 4) and 3 treatment groups (n = 4 each). RG7112, apatinib, and RG7112 + apatinib were administered to the treatment groups and an equal volume of dimethyl sulfoxide to the control group by daily gavage for 14 d. The tumor length (L) and width (W) were measured on alternate days using vernier calipers. The following formula was used to calculate the tumor volume: volume (mm3) = 0.5 × L × W × W. At the end of the experiment, the nude mice were killed by CO2 overdose anesthesia. The tumors were dissected and weighed using a precision balance, and the tumor tissue was stored in liquid nitrogen for further analysis.
item1366 Gemcitabine-resistant Panc1 cells (Panc1-GR) were prepared as stable luciferase clones after transduction with CTRL or shSRSF3 or sh-ANRIL-L vector or SRSF3 or ANRIL-L. For the PDX models, pieces of fresh human pancreatic cancer tissues were transplanted subcutaneously into the axilla of 4-6 week-old NOD/SCID mice.
item1367 To test for malignant transformation, 1×107 cells were inoculated subcutaneously in the dorsal thoracic midline of ten NOD/SCID mice (Weitong Lihua Experimental Animal Technology Co. Ltd). Tumor formation and growth were assessed every 3 days.
item1368 To test for malignant transformation, 1×107 cells were inoculated subcutaneously in the dorsal thoracic midline of ten NOD/SCID mice (Weitong Lihua Experimental Animal Technology Co. Ltd). Tumor formation and growth were assessed every 3 days.
item1369 The male Sprague-Dawley rats (8 weeks old, 200-220 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. Streptozotocin (Sigma, USA) was given by intraperitoneal injection at a dose of 60 mg/Kg to induce diabetics rats, while the control rats were given by empty citrate buffer. One week after induction, those rats with blood glucose levels > 16.7 mmol/L for three times were considered as successful inducted diabetes. All the rats did not receive insulin during the experiments.In the intraocular injection experiments, rats confirmed as the DM model (blood glucose levels > 16.7 mmol/L for three times) were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/Kg). A total of 10 ul DMEM with 1*109 TU lentiviruses (A20-overexpression, OE-A20 group) or the same volume of DMEM with control lentiviruses (OE-NC group) was injected into the vitreous cavity using a 33-gauge needle. This treatment was performed one time per month, and the rats were sacrificed for further experiments at the 3 months.
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item1377 Established a xenograft model in BALB/c nude mice by inoculating HCC827-GR cells transfected with the constructs for circASK1 silencing, ASK1-272a.a overexpression and ASK1-272a.a overexpression/circASK1 knockdown
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item1384 Sixteen BALB/c nude mice (male, 6-week-old) were raised under pathogen-free conditions and randomly divided into two groups. A total of 2 × 106 A375 NTC or sh-METTL3#2 cells were subcutaneously inoculated into the right hind flank. Body weight and tumor size were measured every other day. The tumors were harvested at the end of the observation period, and tumor weight and gross images were recorded (11 days after inoculation). The tumors were embedded in RNA later and 10% formalin for further detection.
item1385 A total of 18 mice were randomly divided into 2 groups, a sham group (n=9), and a MIRI group (n=9). Operators were blinded to the allocation. The MIRI mice were established by ligation of the left anterior descending coronary artery. After peritoneal anesthesia with 1% pentobarbital sodium, surgical incisions were made between the third and fourth sternal intercostals to expose the heart. The left anterior descending coronary artery was identified under the microscope, and the left atrial appendage was ligated at the 1 cm lower margin with a suture needle to induce ischemia for 30 minutes. Then, the slip knot was unwound for 24 h of reperfusion. The sham group was not ligated. No unexpected adverse events were observed and animals were not excluded. Myocardium and peripheral blood were collected after the mice were sacrificed at 24 h reperfusion. Each group was analyzed in at least 3 independent experiments, which made the group size of each experiment 3.
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item1388 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1389 Established a xenograft model in BALB/c nude mice by inoculating HCC827-GR cells transfected with the constructs for circASK1 silencing, ASK1-272a.a overexpression and ASK1-272a.a overexpression/circASK1 knockdown
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item1394 Cells at 1 × 106 were subcutaneously injected into the mice similarly to nude mice. Twenty-eight days later, grafted tumors were collected and morphologically analyzed.
item1395 Cells at 1 × 106 were subcutaneously injected into the mice similarly to nude mice. Twenty-eight days later, grafted tumors were collected and morphologically analyzed.
item1396 .
item1397 KP and Mettl3-/- mice were bred to generate KPM-/- mice. Afterwards, the KP and KPM-/- mice were intranasally infected under anesthesia with adeno-associated virus type 5 (AAV5) expressing Cre to initiate lung tumorigenesis along with ALKBH5-expressing AAV5 or Empty AAV5 to generate KPE, KPA, KPEM-/- and KPAM-/- spontaneous LUAD mouse models. For generation of LLC-based intra-pulmonary tumor mouse models, 1 × 107 LLC cells were injected into C57BL/6 mice via the tail vein.For cell-derived xenograft (CDX) mouse models, 1.0 × 107 H1299 or 1.5 × 107 H1975 cells were subcutaneously injected into 4-6-week-old athymic nude mice. The tumors were monitored at indicated time points and isolated for further analysis after sacrifice.
item1398 KP and Mettl3-/- mice were bred to generate KPM-/- mice. Afterwards, the KP and KPM-/- mice were intranasally infected under anesthesia with adeno-associated virus type 5 (AAV5) expressing Cre to initiate lung tumorigenesis along with ALKBH5-expressing AAV5 or Empty AAV5 to generate KPE, KPA, KPEM-/- and KPAM-/- spontaneous LUAD mouse models. For generation of LLC-based intra-pulmonary tumor mouse models, 1 × 107 LLC cells were injected into C57BL/6 mice via the tail vein.For cell-derived xenograft (CDX) mouse models, 1.0 × 107 H1299 or 1.5 × 107 H1975 cells were subcutaneously injected into 4-6-week-old athymic nude mice. The tumors were monitored at indicated time points and isolated for further analysis after sacrifice.
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item1404 A total of 5 × 106 transfected MKN-45 cells, stably transfected with sh-IGF2BP1 vector or empty vector were subcutaneously injected into the flank of the mice. Tumor growth was measured every three days, and calculated using the following equation = a × b2/2 (a for longitudinal diameter; and b for latitudinal diameter). Three weeks after injection, mice were sacrificed.
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item1408 The mice were randomly divided into control, Ad-sh-NC, and Ad-sh-METTL14 groups (10 mice per group). The mice in the control group were fed a normal diet, while the Ad-sh-NC and Ad-sh-METTL14 groups were fed a high-fat diet (20% fat and 0.25% cholesterol). Furthermore, 300 uL of constructed sh-NC or sh-METTL14 adenovirus was injected every 3 weeks into the caudal veins of mice from the Ad-sh-NC or Ad-sh-METTL14 groups, respectively. The constructed vectors were obtained from HanBio Technology Co., Ltd. (Shanghai, China). All mice were sacrificed after 24 weeks and the aortas were separated for further experiments.
item1409 The mice were randomly divided into control, Ad-sh-NC, and Ad-sh-METTL14 groups (10 mice per group). The mice in the control group were fed a normal diet, while the Ad-sh-NC and Ad-sh-METTL14 groups were fed a high-fat diet (20% fat and 0.25% cholesterol). Furthermore, 300 uL of constructed sh-NC or sh-METTL14 adenovirus was injected every 3 weeks into the caudal veins of mice from the Ad-sh-NC or Ad-sh-METTL14 groups, respectively. The constructed vectors were obtained from HanBio Technology Co., Ltd. (Shanghai, China). All mice were sacrificed after 24 weeks and the aortas were separated for further experiments.
item1410 Female ICR mice with 7 or 8 weeks of age were obtained. To generate the mouse model, according to our previous methods, CTX (Sigma, USA) was used at different doses (40, 80, and 120 mg/kg), CTX with 120 mg/kg was used at different time point, respectively (0, 1, 2, 4, and 8 weeks). The animals were divided into four groups: control (saline injection) and 40, 80, 120 mg/kg CTX treatment groups (n = 10 per group).
item1411 .
item1412 2 × 106 cells resuspended in 50 uL of Matrigel (Corning) were subcutaneously injected into 4-6 weeks old male nude mice. When tumor volumes reached 150-200 mm3, animals were divided into control group and radiotherapy group. In the radiotherapy group, tumors were treated with a single irradiation (4 Gy) when tumor volumes reached approximately 150-200 mm3. The tumor stopped growing in the next few days and then restarted growth.
item1413 2 × 106 cells resuspended in 50 uL of Matrigel (Corning) were subcutaneously injected into 4-6 weeks old male nude mice. When tumor volumes reached 150-200 mm3, animals were divided into control group and radiotherapy group. In the radiotherapy group, tumors were treated with a single irradiation (4 Gy) when tumor volumes reached approximately 150-200 mm3. The tumor stopped growing in the next few days and then restarted growth.
item1414 2 × 106 cells resuspended in 50 uL of Matrigel (Corning) were subcutaneously injected into 4-6 weeks old male nude mice. When tumor volumes reached 150-200 mm3, animals were divided into control group and radiotherapy group. In the radiotherapy group, tumors were treated with a single irradiation (4 Gy) when tumor volumes reached approximately 150-200 mm3. The tumor stopped growing in the next few days and then restarted growth.
item1415 In vivo animal assay, FOXD2-AS1 overexpression promoted the tumor growth in mice subcutaneous injection
item1416 In vivo animal assay, FOXD2-AS1 overexpression promoted the tumor growth in mice subcutaneous injection
item1417 All mice had free access to food and water throughout the study. To test the oncogenic potential of FTO gene, K562 parental cells were transfected with FTO expression or control vectors for 6 h, and 5.0 × 106 transfected cells were subcutaneously injected into the bilateral flanks of the nude mice. For therapeutic experiments, K562 nilotinibR cells (1.5 × 106) were injected subcutaneously into the bilateral flanks of the nude mice. To retain the resistance phenotypes, these mice received 1 mg/kg nilotinib twice a week for 4 weeks till the tumor approached ~10 mm3. Then the tumor-bearing mice were randomly divided into groups of 3 animals and received intraperitoneal injection of either 5 mg/kg nilotinib, 10 mg/kg rhein alone or their combination in polyethylene glycol 400 (PEG400, Sigma #1546445) and saline (ratio 15:38:47), three injections each week for 5 times in total.
item1418 Totally 5 × 106 ESCC cells after treatment were administered to randomized animals by the subcutaneous route (right flank) in 200 uL DMEM. Tumor measurement was performed every other day. At study end (at least 3 weeks), the animals underwent euthanasia, and the tumors were extracted for histology.
item1419 .
item1420 For the xenograft tumor model, approximately 1 × 106 ccRCC cells suspended in 100 uL PBS were subcutaneously inoculated in the right flank of 5-week-old BALB/c nude mice. For the ccRCC orthotopic implantation model, approximately 1 × 106 ccRCC cells suspended in 30 uL Matrigel were injected under the renal capsule of 5-week-old BALB/c nude mice. After 6 weeks, the anesthetized mice were intraperitoneally injected with D-luciferin (Yeason) and imaged using an in vivo imaging system to detect tumor growth and metastasis. For the lung metastasis model, approximately 5 × 105 ccRCC cells suspended in PBS were injected into the tail vein of 5-week-old mice. After 6-8 weeks, mice were anesthetized and lung metastasis was imaged as above.
item1421 For the xenograft tumor model, approximately 1 × 106 ccRCC cells suspended in 100 uL PBS were subcutaneously inoculated in the right flank of 5-week-old BALB/c nude mice. For the ccRCC orthotopic implantation model, approximately 1 × 106 ccRCC cells suspended in 30 uL Matrigel were injected under the renal capsule of 5-week-old BALB/c nude mice. After 6 weeks, the anesthetized mice were intraperitoneally injected with D-luciferin (Yeason) and imaged using an in vivo imaging system to detect tumor growth and metastasis. For the lung metastasis model, approximately 5 × 105 ccRCC cells suspended in PBS were injected into the tail vein of 5-week-old mice. After 6-8 weeks, mice were anesthetized and lung metastasis was imaged as above.
item1422 1×106 MDA-MB-231 cells were resuspended in 100 uL PBS with 50% Matrigel (Corning Costar, USA), and injected into the mammary fat pad of the mice.
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item1430 BALB/c nude mice (5 weeks old) were purchased from the Beijing HFK Bioscience Co. Ltd. 2×106 cells (50 uL) were mixed with 50 uL Matrigel (BD Biosciences, San Jose, CA, USA) were injected subcutaneously in the rear flank fat pad of the nude mice (N = 6, per group).
item1431 BALB/c nude mice (5 weeks old) were purchased from the Beijing HFK Bioscience Co. Ltd. 2×106 cells (50 uL) were mixed with 50 uL Matrigel (BD Biosciences, San Jose, CA, USA) were injected subcutaneously in the rear flank fat pad of the nude mice (N = 6, per group).
item1432 For the subcutaneous xenograft model, PC9 cells stably transfected with METTL3 knockdown (shMETTL3) or negative control (shNC) shRNA (5 × 106 cells per mouse, n = 6) were suspended in 200 uL PBS with 50% Matrigel matrix (Corning, USA, 354234) and then injected into one side of the axilla of nude mice.
item1433 1×106 MDA-MB-231 cells were resuspended in 100 uL PBS with 50% Matrigel (Corning Costar, USA), and injected into the mammary fat pad of the mice.
item1434 Conditional knockout of Fto in bone in mice was generated as previously described. Throughout the study, mice were maintained on a 12 h: 12 h light:dark cycle in a specific pathogen-free facility.
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item1438 A total of 3×106 RPMI8226/MM1R-Luc cells were intravenously injected into NCG mice to establish a disseminated human MM xenograft model. The in vivo antitumor effect of the FTO inhibitor MA2 combined with or without the first-line chemotherapeutic agent BTZ was evaluated as follows: 3 days post xenotransplantation, MA2 (20 mg/kg), or vehicle control was injected intraperitoneally (i.p.) daily for 10 days, and BTZ was injected intraperitoneally on days 1, 4, 8, and 11. Mouse serum was collected at specified time points during the treatment, and the tumor burden was monitored by detecting myeloma cell-secreted Lambda light chains via a Human Lambda ELISA Kit (Bethyl Laboratories, No. E88-116). Tumor development was monitored weekly after treatment with an in vivo imaging system (IVIS, SI Imaging, Lago, and LagoX). Luciferin (150 mg/kg, YEASEN, Shanghai, China) was injected intraperitoneally into the mice.
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item1445 KOV-3 cells (1 ×106) stable transfected with METTL14 or control lentivirus, were injected subcutaneously into the right flank of BALB/c nude mice.
item1446 For mouse xenograft tumor model, 1×106 Saos2 or HOS cells were mixed in 200 ul PBS and injected subcutaneously into 6- to 8-week-old C57BL/6 mice.umor size of mice was measured by vernier calipers every week after injection. The mice were euthanized after 28 days by intraperitoneal injection of 1% pentobarbital sodium (120 mg/kg), and the tumor tissues were removed from the mice.
item1447 Five-week-old male nude BALB/C mice were applied for this animal studies and fed with certified standard diet and tap water ad libitum in a light/dark cycle of 12 h on/12 h off.The assay was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Stable transfection of METTL3 knockdown (sh-METTL3) or negative control (sh-blank) in SiHa cells (5 × 106 cells per 0.1 mL) were injected into the flank of mice.
item1448 Five-week-old male nude BALB/C mice were applied for this animal studies and fed with certified standard diet and tap water ad libitum in a light/dark cycle of 12 h on/12 h off.The assay was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Stable transfection of METTL3 knockdown (sh-METTL3) or negative control (sh-blank) in SiHa cells (5 × 106 cells per 0.1 mL) were injected into the flank of mice.
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item1453 Mice were divided into two groups (n = 4/group) randomly. 3×106 cells suspended in 200 uL PBS were administered via subcutaneous injection over the right flank region of nude mice. After the development of palpable tumors (average volume, 50 mm3), intratumoral injection of synthetic miR-193b, or negative control complexed with siPORT Amine transfection reagent (Ambion, USA) was given 6 times at a 4-day interval.
item1454 Mice were divided into two groups (n = 4/group) randomly. 3×106 cells suspended in 200 uL PBS were administered via subcutaneous injection over the right flank region of nude mice. After the development of palpable tumors (average volume, 50 mm3), intratumoral injection of synthetic miR-193b, or negative control complexed with siPORT Amine transfection reagent (Ambion, USA) was given 6 times at a 4-day interval.
item1455 Established subcutaneous xenografts in athymic nude mice with MIR100HGKOE4 CC-CR cells transduced with a luciferase-expressing lentiviral vector and then treated the mice with cetuximab.
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item1459 PC9-GR cells stably infected with KIAA1429-targeting shRNA and control were suspended in 100 uL of PBS with Matrigel matrix (BD Biosciences). Then, cells were injected into one of the flanks of BALB/c nude mice.
item1460 Four-week-old BALB/c nude mice were randomly divided into three groups: (1) vector group, (2) vector + Bete-elemene group, and (3) Bete-elemene + METTL3 group. Nude mice were raised in an SPF level animal house and were free to eat and drink. Mice in the vector group were subcutaneously injected with lung cancer cells transfected with empty vector and did not receive Bete-elemene administration, and this group was implemented as the negative control. Following establishing orthotopic xenografts by using A549 or H1299 cells transfected with empty vector, mice in the vector + Bete-elemene group underwent intraperitoneal injection with Bete-elemene once a day. For the subcutaneous transplanted model, A549 or H1299 cells transfected with METTL3-overexpressing vector were inoculated into mice from the Bete-elemene + METTL3 group. Then, mice were intraperitoneally administrated with Bete-elemene once a day. Three weeks later, all the animals were euthanized with CO2. Xenografts were removed and weighted after mice were euthanatized.
item1461 .
item1462 .
item1463 Six-week-old male BALB/c nude mice were maintained under specific pathogen-free conditions,Mice were randomly assigned to 2 groups (N = 6). In each group, lentiviral-transduced SK-Cha-1 cells (2.5 × 106) were subcutaneously injected into the dorsal right flanks of the mice, and the mice were monitored every 3 days for tumor growth.
item1464 .
item1465 .
item1466 Established subcutaneous xenografts in athymic nude mice with MIR100HGKOE4 CC-CR cells transduced with a luciferase-expressing lentiviral vector and then treated the mice with cetuximab.
item1467 .
item1468 .
item1469 .
item1470 Implanting 5000 human derived GSCs into the right cerebral cortex of NSG mice at a depth of 3.5 mm under a University of California, San Diego Institutional Animal Care and Use Committee (IACUC) approved protocol. Brains were harvested and fixed in 4% formaldehyde, cryopreserved in 30% sucrose, and then cryosectioned. Hematoxylin and eosin (H&E) staining was performed on sections for histological analysis. In parallel survival experiments, mice were observed until the development of neurological signs. For in vivo drug treatment studies, intracranial xenografts were generated by implanting 5000 patient-derived GSCs (387 and 4121) into the right cerebral cortex of NSG mice as described above. Mice recovered for 7 days were randomly assigned into drug vs. treatment group by a blinded investigator. Mice were then treated daily with either vehicle (25 mM Tartaric acid) or 50 mg/kg linsitinib by oral gavage.
item1471 For induction of ESCC, 4-week-old mice were treated with drinking water containing 50 ug/mL 4NQO (Sigma-Aldrich, USA) for 16 weeks and then given normal drinking water for another 4-5 weeks. Cre was activated by the intraperitoneal injection of tamoxifen (Sigma-Aldrich, USA) at a dose of 9 mg per 40 g body weight every other day for a total of three injections. For tumor measurement, mice were sacrificed, and the esophagus was dissected immediately. The surface areas of tumors were measured as described previously.
item1472 .
item1473 .
item1474 For subcutaneous xenograft experiments in B-NDG mice, approximately 1 × 106 MDA-MB-231 and there was subcutaneous injection of the cells that resuspended in 100 uL PBS into the left flank of the mice and were divided into 11 groups randomly (each containing 5 mice). After the treatment Atezolizumab (Selleck, Shanghai, China) or corresponding iso control antibody (Selleck, Shanghai, China) was injected intratumorally on day 3, 6, 9, 12, 15 post-MDA-MB-231 inoculations, and 5 × 106 cytokine-induced killer (CIK) cells were injected in the tail vein on day 7, 14, 21.
item1475 For subcutaneous xenograft experiments in B-NDG mice, approximately 1 × 106 MDA-MB-231 and there was subcutaneous injection of the cells that resuspended in 100 uL PBS into the left flank of the mice and were divided into 11 groups randomly (each containing 5 mice). After the treatment Atezolizumab (Selleck, Shanghai, China) or corresponding iso control antibody (Selleck, Shanghai, China) was injected intratumorally on day 3, 6, 9, 12, 15 post-MDA-MB-231 inoculations, and 5 × 106 cytokine-induced killer (CIK) cells were injected in the tail vein on day 7, 14, 21.
item1476 .
item1477 1×106 MDA-MB-231 cells were resuspended in 100 uL PBS with 50% Matrigel (Corning Costar, USA), and injected into the mammary fat pad of the mice.
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item1488 1×106 MDA-MB-231 cells were resuspended in 100 uL PBS with 50% Matrigel (Corning Costar, USA), and injected into the mammary fat pad of the mice.
item1489 .
item1490 .
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item1492 .
item1493 .
item1494 Liver fibrosis mice model was induced by intraperitoneal CCl4 injection for 12 weeks. The dose regimen for CCl4 in mice is 1 ml kg-1, diluted to 50% with olive oil twice per week for 12 weeks. Until 11 weeks, mice with liver-specific disruption of ALKBH5 were given hydrodynamic tail-vein injections of LV5-ALKBH5.
item1495 Mettl3 floxed mice were purchased from GemPharmatech Co. Ltd (Nanjing, China). Pdgfr-Beta-Cre mice were purchased from Beijing Biocytogen Co. Ltd (Beijing, China) generated on C57BL/6J background. Mettl3 flox/flox mice were crossed with Pdgfr-Beta-Cre mice to generate pericyte-specific Mettl3 knockout mice. All mice were bred under the specific-pathogen free condition with free access to diet and water or their nursing mothers with alternating 12/12 light-dark cycle (lights on at 08:00 and off at 20:00).
item1496 Mettl3 floxed mice were purchased from GemPharmatech Co. Ltd (Nanjing, China). Pdgfr-Beta-Cre mice were purchased from Beijing Biocytogen Co. Ltd (Beijing, China) generated on C57BL/6J background. Mettl3 flox/flox mice were crossed with Pdgfr-Beta-Cre mice to generate pericyte-specific Mettl3 knockout mice. All mice were bred under the specific-pathogen free condition with free access to diet and water or their nursing mothers with alternating 12/12 light-dark cycle (lights on at 08:00 and off at 20:00).
item1497 Mettl3 floxed mice were purchased from GemPharmatech Co. Ltd (Nanjing, China). Pdgfr-Beta-Cre mice were purchased from Beijing Biocytogen Co. Ltd (Beijing, China) generated on C57BL/6J background. Mettl3 flox/flox mice were crossed with Pdgfr-Beta-Cre mice to generate pericyte-specific Mettl3 knockout mice. All mice were bred under the specific-pathogen free condition with free access to diet and water or their nursing mothers with alternating 12/12 light-dark cycle (lights on at 08:00 and off at 20:00).
item1498 The nude mice were maintained under pathogen-free conditions and kept under timed lighting conditions mandated by the committee with food and water provided ad libitum. For xenograft experiments, nude mice were injected subcutaneously with 5 × 106 cells resuspended in 0.1 mL PBS. When a tumor was palpable, it was measured every 3 days.
item1499 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1500 .
item1501 The nude mice were maintained under pathogen-free conditions and kept under timed lighting conditions mandated by the committee with food and water provided ad libitum. For xenograft experiments, nude mice were injected subcutaneously with 5 × 106 cells resuspended in 0.1 mL PBS. When a tumor was palpable, it was measured every 3 days.
item1502 METTL14+/- mice are generated by mating wild-type mice (C57/BL6 background) with METTL14+/- mice. METTL14+/-/APOE-/- healthy offspring mice are produced by heterozygous METTL14+/- mice and heterozygous APOE-/- mice by Mendelian ratios. APOE-/- mice and C57/BL6 mice were purchased from Model Animal Research Center of Nanjing (Nanjing, Jiangsu, China). All mice were housed in the Laboratory Animals Center of the Henan Provincial People's Hospital, with controlled temperature and humidity and a 12:12-hour dark-light cycle, and were provided water and mouse chow ad libitum.
item1503 Mettl14-/+ mice are generated by mating wild-type mice (C57/BL6 background) with Mettl14-/+ mice. Mettl14-/+/APOE-/- healthy offspring mice are produced by heterozygous Mettl14-/+ mice and heterozygous APOE-/- mice by Mendelian ratios. APOE-/- mice and C57/BL6 mice were purchased from Model Animal Research Center of Nanjing (Nanjing, Jiangsu, China). All mice were housed in the Laboratory Animals Center of the Henan Provincial People's Hospital, with controlled temperature and humidity and a 12:12-hour dark-light cycle, and were provided water and mouse chow ad libitum.
item1504 .
item1505 .
item1506 Approximately 2 × 106 PCa cells (PC-3 shNC, shYTHDF2, shMETTL3 cell lines) per mouse suspended in 100 uL PBS were injected in the flank of male BALB/c nude mice (4 weeks old). During the 40-day observation, the tumor size (V = (width2×length ×0.52)) was measured with vernier caliper. Approximately 1.5 × 106 PCa cells suspended in 100 uL of PBS (PC-3 shNC, shYTHDF2, and shMETTL3 cell lines) per mouse were injected into the tail vein of male BALB/c nude mice (4 weeks old). The IVIS Spectrum animal imaging system (PerkinElmer) was used to evaluate the tumor growth (40 days) and whole metastasis conditions (4 weeks and 6 weeks) with 100 uL XenoLight D-luciferin Potassium Salt (15 mg/ml, Perkin Elmer) per mouse. Mice were anesthetized and then sacrificed for tumors and metastases which were sent for further organ-localized imaging as above, IHC staining and hematoxylin-eosin (H&E) staining.
item1507 Approximately 2 × 106 PCa cells (PC-3 shNC, shYTHDF2, shMETTL3 cell lines) per mouse suspended in 100 uL PBS were injected in the flank of male BALB/c nude mice (4 weeks old). During the 40-day observation, the tumor size (V = (width2×length ×0.52)) was measured with vernier caliper. Approximately 1.5 × 106 PCa cells suspended in 100 uL of PBS (PC-3 shNC, shYTHDF2, and shMETTL3 cell lines) per mouse were injected into the tail vein of male BALB/c nude mice (4 weeks old). The IVIS Spectrum animal imaging system (PerkinElmer) was used to evaluate the tumor growth (40 days) and whole metastasis conditions (4 weeks and 6 weeks) with 100 uL XenoLight D-luciferin Potassium Salt (15 mg/ml, Perkin Elmer) per mouse. Mice were anesthetized and then sacrificed for tumors and metastases which were sent for further organ-localized imaging as above, IHC staining and hematoxylin-eosin (H&E) staining.
item1508 Approximately 2 × 106 PCa cells (PC-3 shNC, shYTHDF2, shMETTL3 cell lines) per mouse suspended in 100 uL PBS were injected in the flank of male BALB/c nude mice (4 weeks old). During the 40-day observation, the tumor size (V = (width2×length ×0.52)) was measured with vernier caliper. Approximately 1.5 × 106 PCa cells suspended in 100 uL of PBS (PC-3 shNC, shYTHDF2, and shMETTL3 cell lines) per mouse were injected into the tail vein of male BALB/c nude mice (4 weeks old). The IVIS Spectrum animal imaging system (PerkinElmer) was used to evaluate the tumor growth (40 days) and whole metastasis conditions (4 weeks and 6 weeks) with 100 uL XenoLight D-luciferin Potassium Salt (15 mg/ml, Perkin Elmer) per mouse. Mice were anesthetized and then sacrificed for tumors and metastases which were sent for further organ-localized imaging as above, IHC staining and hematoxylin-eosin (H&E) staining.
item1509 Approximately 2 × 106 PCa cells (PC-3 shNC, shYTHDF2, shMETTL3 cell lines) per mouse suspended in 100 uL PBS were injected in the flank of male BALB/c nude mice (4 weeks old). During the 40-day observation, the tumor size (V = (width2×length ×0.52)) was measured with vernier caliper. Approximately 1.5 × 106 PCa cells suspended in 100 uL of PBS (PC-3 shNC, shYTHDF2, and shMETTL3 cell lines) per mouse were injected into the tail vein of male BALB/c nude mice (4 weeks old). The IVIS Spectrum animal imaging system (PerkinElmer) was used to evaluate the tumor growth (40 days) and whole metastasis conditions (4 weeks and 6 weeks) with 100 uL XenoLight D-luciferin Potassium Salt (15 mg/ml, Perkin Elmer) per mouse. Mice were anesthetized and then sacrificed for tumors and metastases which were sent for further organ-localized imaging as above, IHC staining and hematoxylin-eosin (H&E) staining.
item1510 Approximately 2 × 106 PCa cells (PC-3 shNC, shYTHDF2, shMETTL3 cell lines) per mouse suspended in 100 uL PBS were injected in the flank of male BALB/c nude mice (4 weeks old). During the 40-day observation, the tumor size (V = (width2×length ×0.52)) was measured with vernier caliper. Approximately 1.5 × 106 PCa cells suspended in 100 uL of PBS (PC-3 shNC, shYTHDF2, and shMETTL3 cell lines) per mouse were injected into the tail vein of male BALB/c nude mice (4 weeks old). The IVIS Spectrum animal imaging system (PerkinElmer) was used to evaluate the tumor growth (40 days) and whole metastasis conditions (4 weeks and 6 weeks) with 100 uL XenoLight D-luciferin Potassium Salt (15 mg/ml, Perkin Elmer) per mouse. Mice were anesthetized and then sacrificed for tumors and metastases which were sent for further organ-localized imaging as above, IHC staining and hematoxylin-eosin (H&E) staining.
item1511 .
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item1518 The rats were randomly divided into a fresh air group and a COPD group and each group contained 10 rats. The COPD group was placed into a breathable cage and exposed to PM2.5 (1.46 mg/m3) from the motor vehicle exhaust for two 2-h exposure periods (from 9:00 a.m. to 11:00 a.m. and from 15:00 p.m. to 17:00 p.m.), 5 days per week, for 7 months. After each period of exposure, the animals were exposed to fresh air for 30 min to take a rest. Gasoline-powered motorcycle used as of source of the Particulate matter (PM) pollution was located next door to the rat exposure room, and the PM was directed towards the exposure room through a metal tube. Fresh air was sent to through an air pump. After exposed for 7 months, rats were performed lung function test using a Forced Pulmonary Maneuver System (Buxco Research Systems, Wilmington, NC, USA).
item1519 Nude mice (4 weeks, male) were used for tumor model.For the subcutaneous tumor model, 1 × 106 shNC, shWTAP or shHMBOX1 or shWTAP/shHMBOX1 U2OS cells seeded into mice via subcutaneous injection. Tumor volume and tumor weight were measured to analyze tumor growth as previous described. For orthotopic xenograft tumor model, shNC, shWTAP, shHMBOX1, or shWTAP/shHMBOX1 U2OS cells were labeled with a luminescent dye and GFP, and injected into the cavity of the tibia of mice. Thirty days after injection, tumor growth was detected. For the metastasis model, the cells were injected into the tail vein, and the lung metastasis were detected 30 days after injection using in vivo bioluminescence imaging system.
item1520 BALB/c male nude mice were 4 weeks old. GBM cells with stable overexpression or knockdown of FTO and ovNC or shNC were transduced with lentivirus expressing luciferase. The cells were intracranially injected at a density of 5 × 105/10 uL into every mouse to form an orthotopic xenograft model. Coordinates of injection were 1 mm anterior and 2.5 mm right to the bregma, at a depth of 3.5 mm (the right frontal lobes of the mouse). Every 6 days, bioluminescence imaging (IVIS Lumina Series III; PerkinElmer, Waltham, MA) was used to image the mouse. At 8 days, we randomly chose 5 mice from each group to euthanize them, and their brain tissues were fixed with paraformaldehyde for further study. Another 5 mice were used for survival time analysis. For DB2313 (563801; MedKoo) anti-tumor research, male nude mice were subcutaneously injected with 5 × 106 U87MG cells suspended in 0.1 mL PBS. After 7 days, mice were intraperitoneally injected with DB2313 at density of 10 mg/kg/day dissolved in PBS solvent containing 10% DMSO for 7 days. The other group treated with vehicle only was set as the control group.
item1521 BALB/c male nude mice were 4 weeks old. GBM cells with stable overexpression or knockdown of FTO and ovNC or shNC were transduced with lentivirus expressing luciferase. The cells were intracranially injected at a density of 5 × 105/10 uL into every mouse to form an orthotopic xenograft model. Coordinates of injection were 1 mm anterior and 2.5 mm right to the bregma, at a depth of 3.5 mm (the right frontal lobes of the mouse). Every 6 days, bioluminescence imaging (IVIS Lumina Series III; PerkinElmer, Waltham, MA) was used to image the mouse. At 8 days, we randomly chose 5 mice from each group to euthanize them, and their brain tissues were fixed with paraformaldehyde for further study. Another 5 mice were used for survival time analysis. For DB2313 (563801; MedKoo) anti-tumor research, male nude mice were subcutaneously injected with 5 × 106 U87MG cells suspended in 0.1 mL PBS. After 7 days, mice were intraperitoneally injected with DB2313 at density of 10 mg/kg/day dissolved in PBS solvent containing 10% DMSO for 7 days. The other group treated with vehicle only was set as the control group.
item1522 .
item1523 .
item1524 Mettl3fl/wt mice were generated as previously described. Mettl3fl/wt mice were crossed with K14CreER mice to obtain K14creER/Mettl3fl/fl (cKO) mice. Mettl3 cKO and control mice were injected with tamoxifen and then were subjected to corneal alkali burn treatment. The right eye was the experimental eye, and the left eye was the control eye. The mice were sacrificed at 24 hours, 7 days, 14 days, 35 days, and 56 days after injury. Six mice were taken from each period. Both eyes were removed, frozen in OCT (n = 4), fixed in 4% paraformaldehyde, and embedded in conventional paraffin (n = 2).
item1525 Mettl3fl/wt mice were generated as previously described. Mettl3fl/wt mice were crossed with K14CreER mice to obtain K14creER/Mettl3fl/fl (cKO) mice. Mettl3 cKO and control mice were injected with tamoxifen and then were subjected to corneal alkali burn treatment. The right eye was the experimental eye, and the left eye was the control eye. The mice were sacrificed at 24 hours, 7 days, 14 days, 35 days, and 56 days after injury. Six mice were taken from each period. Both eyes were removed, frozen in OCT (n = 4), fixed in 4% paraformaldehyde, and embedded in conventional paraffin (n = 2).
item1526 BALB/C nude mice (5 weeks old, weighing 18-22 g) were fed in specific pathogen-free facilities and subcutaneously inoculated with U266 cells (1 × 106). The mice were randomly divided into 3 groups with 6 mice per group, when the tumor was measurable. Then, miR-27a-3p mimic or sh-METTL3 was injected intratumorally at an interval of 4 days a total of 4 times. Tumor volume was measured using a digital caliper every week and calculated using the formula V = 1/2 (width2 × length).
item1527 BALB/C nude mice (5 weeks old, weighing 18-22 g) were fed in specific pathogen-free facilities and subcutaneously inoculated with U266 cells (1 × 106). The mice were randomly divided into 3 groups with 6 mice per group, when the tumor was measurable. Then, miR-27a-3p mimic or sh-METTL3 was injected intratumorally at an interval of 4 days a total of 4 times. Tumor volume was measured using a digital caliper every week and calculated using the formula V = 1/2 (width2 × length).
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item1539 Mettl14 heterozygous mice (Mettl14-/+) were established from C57/BL6 mice by Cyagen Biosciences, Inc. (Suzhou, Jiangsu, China), using CRISPR/Cas9-based targeting and homology-directed repair. C57/BL6 and APOE-/- mice were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Mettl14-/+APOE-/- mice were generated by breeding Mettl14-/+ mice with APOE-/- mice. Eight- to 10-week-old male APOE-/- (WT) mice and Mettl14-/+APOE-/- (KO) mice were fed a high-cholesterol diet (D12108C, Opensource diets) for 12 weeks. Then, the mice were euthanized for further analysis.
item1540 Mettl14 heterozygous mice (Mettl14-/+) were established from C57/BL6 mice by Cyagen Biosciences, Inc. (Suzhou, Jiangsu, China), using CRISPR/Cas9-based targeting and homology-directed repair. C57/BL6 and APOE-/- mice were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Mettl14-/+APOE-/- mice were generated by breeding Mettl14-/+ mice with APOE-/- mice. Eight- to 10-week-old male APOE-/- (WT) mice and Mettl14-/+APOE-/- (KO) mice were fed a high-cholesterol diet (D12108C, Opensource diets) for 12 weeks. Then, the mice were euthanized for further analysis.
item1541 .
item1542 male C57BL/6J mice (6 weeks old) were given drinking water containing 0.05% (w/v) BBN (TCI, catalog no. B0938) for 20 weeks. After the BBN administration, mice were given normal drinking water and injected with 10% DMSO (as a control) or 20 mg/kg SP600125 (Selleck, catalog no. 129-56-6) i.p. every 3 days. After seven injections, mice were euthanized for tissue retrieval. For the bladder cancer cell-derived xenograft mouse model, male C57BL/6J mice (6 weeks old) were injected subcutaneously with 1 × 106 MB49 cells. One week after bladder cancer cell injection, 10% DMSO or 20 mg/kg SP600125 were injected i.p. every 3 days.
item1543 Approximately 5×106 normal H1299 cells or stable YTHDC2-overexpressing H1299 cells were implanted subcutaneously into the right flank of the animals (n=8 mice per group). Animals were euthanized by cervical dislocation ~30 days after implantation, and tumors were collected and photographed.
item1544 .
item1545 .
item1546 2 × 107 PARP inhibitor resistant PEO1 cells were suspended in 200 uL PBS : Matrigel (1:1) unilaterally injected subcutaneously into the right dorsal flank of 6-8 week-old female immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD/SCID) gamma (NSG) mice. When the average tumor size reached ~100 mm3, the mice were then randomized into four groups and treated with vehicle control, Olaparib (50 mg/kg), XAV939 (5 mg/kg) or a combination daily for 18 days.
item1547 2 × 107 PARP inhibitor resistant PEO1 cells were suspended in 200 uL PBS : Matrigel (1:1) unilaterally injected subcutaneously into the right dorsal flank of 6-8 week-old female immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD/SCID) gamma (NSG) mice. When the average tumor size reached ~100 mm3, the mice were then randomized into four groups and treated with vehicle control, Olaparib (50 mg/kg), XAV939 (5 mg/kg) or a combination daily for 18 days.
item1548 An incision was made in the skin along the medial dorsal line to the aponeurotic and muscular planes, and the posterior vertebral arches were exposed from T8 to T12. Under the dissection stereomicroscope, 3-mm-long laminectomy was performed on the caudal end of T10 vertebra and the rostral end of T11 vertebra. The Infinite Horizons impactor (Infinite Horizons, L.L.C., Lexington, KY, USA) was adopted to produce the contusion SCI using a force of 60 kdyn/cm2. The SCI model rats were established and randomly assigned to SCI model group, ant-NC (negative control, SCI rats treated with lentiviral (lv)-shRNA NC of Mettl14) group and ant-Mettl14 group (SCI rats treated with lv-shRNA of Mettl14). Rats were subjected to laminectomy and then treated with lv-shRNA Mettl14/lv-shRNA-NC (50 ul/day, 100 nmoL/mL; RiboBio, Guangzhou, China) via an intrathecal injection through lumbar puncture for 3 days (0, 1, and 2 days) after 15 min of SCI modelling. In addition, the unmodeled rats were set as sham group.
item1549 An incision was made in the skin along the medial dorsal line to the aponeurotic and muscular planes, and the posterior vertebral arches were exposed from T8 to T12. Under the dissection stereomicroscope, 3-mm-long laminectomy was performed on the caudal end of T10 vertebra and the rostral end of T11 vertebra. The Infinite Horizons impactor (Infinite Horizons, L.L.C., Lexington, KY, USA) was adopted to produce the contusion SCI using a force of 60 kdyn/cm2. The SCI model rats were established and randomly assigned to SCI model group, ant-NC (negative control, SCI rats treated with lentiviral (lv)-shRNA NC of Mettl14) group and ant-Mettl14 group (SCI rats treated with lv-shRNA of Mettl14). Rats were subjected to laminectomy and then treated with lv-shRNA Mettl14/lv-shRNA-NC (50 ul/day, 100 nmoL/mL; RiboBio, Guangzhou, China) via an intrathecal injection through lumbar puncture for 3 days (0, 1, and 2 days) after 15 min of SCI modelling. In addition, the unmodeled rats were set as sham group.
item1550 .
item1551 For induction of BCa, 6-8-week-old mice were treated with drinking water containing 500 ug/ml BBN for 16 weeks and then given normal water for another 10 weeks. Tamoxifen was intraperitonelly injected to the mice with 0.08 mg/g of body weight each day for 3 days in order to inductively knock out the target gene.
item1552 For induction of BCa, 6-8-week-old mice were treated with drinking water containing 500 ug/ml BBN for 16 weeks and then given normal water for another 10 weeks. Tamoxifen was intraperitonelly injected to the mice with 0.08 mg/g of body weight each day for 3 days in order to inductively knock out the target gene.
item1553 They were subcutaneously and caudal vein injected with YTHDC2 knockout AGS cells, respectively. After 7 weeks, the mice were sacrificed and tumor size and lung metastasis nodules were recorded.
item1554 A549 cells were transfected with lentivirus-packaged sh-HNRNPA2B1 (lv-sh-HNRNPA2B1) or control (lv-shCtrl). Then, each mouse was injected subcutaneously with A549 cells of indicated transfection group to generate xenografts. The tumor volume ((width2 × length)/2) was evaluated 4 days a time until 28 days.
item1555 .
item1556 For the subcutaneous transplantation model, 100 uL of 1 × 106 cells were injected subcutaneously into the right armpit of BALB/c nude mice. Animal weight and tumor diameter were measured once a week from the time of implantation.For the pancreatic cancer orthotopic implantation model, 200 uL of Panc02-lucifer cells (2 × 107) were injected into the pancreas in mice anesthetized and laparotomized. After 4 weeks, the mice were anesthetized and injected with 150 mg/kg d-luciferin, via the tail vein.For the liver metastasis model, BALB/c nude mice received 2 × 106 cells (in 100 uL DMEM), directly injected into the spleen. Their body weight was measured once a week from the time of implantation.
item1557 .
item1558 After 7 days of acclimatization, STZ, dissolved in 0.1 M citrate buffer, was intraperitoneally administered daily to mice at a dose of 50 mg/kg after 12 h of food deprivation each day for 5 consecutive days. The type 1 mice diabetic mice were randomly separated into four groups (n = 5-8): AAV9-scramble-control group; AAV9-scramble-STZ; AAV9-shRNA-control; and AAV9-shRNA-STZ group (Hanbio Biotechnology, China). The 50 UL titer of 1 × 1012 virus was injected into the renal pelvis using an insulin needle and maintained there for 2 to 3 s. The type 2 diabetic mice were randomly separated into four groups (n = 5-8): AAV9-scramble-db/m group; AAV9-scramble-db/db group; AAV9-shRNA-db/m group; and AAV9-shRNA-db/db group (Hanbio Biotechnology, China). The 100 UL titer of 1 × 1012 virus was injected into the tail vein using an insulin needle and maintained there for 2 to 3 s. Blood glucose was monitored weekly in mice. At the end of 12 weeks, the 24-h urine samples were collected from the mice kept in metabolic cages.
item1559 A total of 40 BALB/c nude mice were chosen and assigned to two groups: shCtrl group (injected with HepG2 cells) and shIGF2BP2 group (injected with HepG2 cells with IGF2BP2 knockdown). 200 ul of the above cell suspension containing 2 × 105 cells was injected into the left or right back of each mice.
item1560 Each group included 3 mice. 1.0?×?106 stably transfected ACHN cells in 150 uL were injected into a tail vein of each mouse, 45 days after which lungs were excised from the sacrificed mice and stained by Hematoxylin and Eosin (HE) Staining.
item1561 About 1 × 107 stable METTL3 overexpression and negative control SUM-1315 cells were injected subcutaneously into the axilla of the female BALB/C nude mice (4-6 weeks old, 18-20 g, 10 mice/group). One week after injection, the two groups, METTL3 OE and NC, were then randomly allocated into the control group and experimental group (5 mice/group), which were treated with PBS or metformin (250 mg/kg/dose, respectively). PBS or metformin was administered every 2 days via intraperitoneal injection.
item1562 Male C57BL/6J mice were anesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg). The abdominal aorta between the renal arteries and the bifurcation of the iliac arteries was disassociated from the surrounding structures. Video microscopy was used to assay the diameter of the aorta in triplicate. After the measurements were taken, a small piece of gauze dipped in 0.5 mol/L CaCl2 was spread perivascularly onto the aortic passage for 15 min. Control mice received substitute treatment with NaCl (0.9%)-soaked gauze for 15 min. Then, the aorta was rinsed with 0.9% sterile saline, and the incision was sutured. After 3 or 6 weeks, all the animals were sacrificed, and the aortas were harvested for further analysis.
item1563 The mice were divided into two groups with three mice in each group and their flank was subcutaneously injected with 1 × 107 OS cells. 28 days following subcutaneous injection, the mice were sacrificed through carbon dioxide euthanasia (30%/min) to obtain tumor weight and volume measurements.
item1564 Eighteen BALB/C female nude mice aged 4-5 weeks and weighing 15-18 g were randomly assigned into three groups of six mice. The MCF-7 cell lines stably transfected with sh-NC + oe-NC, sh-METTL3 + oe-NC and sh-METTL3 + oe-HMGA2 were selected for subcutaneous establishment of the BC cell line MCF-7 as xenografts in the nude mice. For this purpose, MCF-7 cell lines in the logarithmic growth stage were prepared into a suspension with a concentration of about 1 × 107 cells/ml. The prepared cell suspension was injected into the left armpit of the mice, and the subsequent tumor growth was recorded.
item1565 Eighteen BALB/C female nude mice aged 4-5 weeks and weighing 15-18 g were randomly assigned into three groups of six mice. The MCF-7 cell lines stably transfected with sh-NC + oe-NC, sh-METTL3 + oe-NC and sh-METTL3 + oe-HMGA2 were selected for subcutaneous establishment of the BC cell line MCF-7 as xenografts in the nude mice. For this purpose, MCF-7 cell lines in the logarithmic growth stage were prepared into a suspension with a concentration of about 1 × 107 cells/ml. The prepared cell suspension was injected into the left armpit of the mice, and the subsequent tumor growth was recorded.
item1566 .
item1567 Eighteen BALB/C female nude mice aged 4-5 weeks and weighing 15-18 g were randomly assigned into three groups of six mice. The MCF-7 cell lines stably transfected with sh-NC + oe-NC, sh-METTL3 + oe-NC and sh-METTL3 + oe-HMGA2 were selected for subcutaneous establishment of the BC cell line MCF-7 as xenografts in the nude mice. For this purpose, MCF-7 cell lines in the logarithmic growth stage were prepared into a suspension with a concentration of about 1 × 107 cells/ml. The prepared cell suspension was injected into the left armpit of the mice, and the subsequent tumor growth was recorded.
item1568 5.0 × 106 SW480 cells (infected with scr or KIAA1429 shRNA) that suspended in 50 ul PBS and mixed with an equal volume of matrigel were subcutaneously injected in a 6-weeks-old male NOD/SCID (The Jackson Laboratory, Stock No: 001303) mice flank. We started measuring tumor size at the indicated times one week after injection.
item1569 Mettl5 +/- mice with C57BL/6N background were generated by using CRISPR-Cas9 systems. The exon 2, exon 3 and exon 4 of Mettl5 were deleted in the knockout Mettl5 allele. Mice were genotyped for the targeted allele by PCR using tail DNA.
item1570 Mice were anesthetized with 0.3% sodium pentobarbital (75 mg×kg-1) intraperitoneally, and the aortic arch was tied with a 6-0 nylon suture between the brachiocephalic and left common artery with a homemade L-shaped 26G cushion needle. After ligation, the needle was quickly removed, and the skin was closed. The sham operation was identical, except that the thread was not ligated. Moreover, mice were injected with rAAV9 (4 × 1011 vector genomes (vg)/mouse) carrying an empty vector, YTHDF2 or YTH-del via the tail vein.
item1571 .
item1572 HCC-LM3 cells transfected with sh-NC and sh-RBM15B-3 were injected into the axilla or tail vein of mice.
item1573 5 × 106 A549 cells overexpressing METTL3 (Lv-METTL3) or control (Lv-Ctrl) were suspended in 100?uL phosphate-buffered saline (PBS), and were subcutaneously injected into mouse lower right flank. Drug treatment started in the Lv-METTL3 group when the tumour volume reached around 100 mm3. Mice were randomly divided into three groups to receive vehicle, GSK2536771 (30 mg/kg) or rapamycin (1 mg/kg). Drugs were administrated daily through intraperitoneal injection for 18 days. Treatment conditions were chosen as previously reported.
item1574 5 × 106 A549 cells overexpressing METTL3 (Lv-METTL3) or control (Lv-Ctrl) were suspended in 100?uL phosphate-buffered saline (PBS), and were subcutaneously injected into mouse lower right flank. Drug treatment started in the Lv-METTL3 group when the tumour volume reached around 100 mm3. Mice were randomly divided into three groups to receive vehicle, GSK2536771 (30 mg/kg) or rapamycin (1 mg/kg). Drugs were administrated daily through intraperitoneal injection for 18 days. Treatment conditions were chosen as previously reported.
item1575 5 × 106 A549 cells overexpressing METTL3 (Lv-METTL3) or control (Lv-Ctrl) were suspended in 100?uL phosphate-buffered saline (PBS), and were subcutaneously injected into mouse lower right flank. Drug treatment started in the Lv-METTL3 group when the tumour volume reached around 100 mm3. Mice were randomly divided into three groups to receive vehicle, GSK2536771 (30 mg/kg) or rapamycin (1 mg/kg). Drugs were administrated daily through intraperitoneal injection for 18 days. Treatment conditions were chosen as previously reported.
item1576 .
item1577 .
item1578 .
item1579 .
item1580 .
item1581 Once the tumor volume increased to about 1 cm3, six groups of MCF7 bearing mice (n = 10 in each group) were injected with PBS (0.1 ml, caudal vein) and adriamycin (0.1 ml, 10 mg/kg), respectively. When the tumor reached 1.5 cm in any direction (defined as event-free survival analysis), 10 mice in each group were selected to measure the tumor size and weight on the 12th day after adriamycin injection.
item1582 Once the tumor volume increased to about 1 cm3, six groups of MCF7 bearing mice (n = 10 in each group) were injected with PBS (0.1 ml, caudal vein) and adriamycin (0.1 ml, 10 mg/kg), respectively. When the tumor reached 1.5 cm in any direction (defined as event-free survival analysis), 10 mice in each group were selected to measure the tumor size and weight on the 12th day after adriamycin injection.
item1583 Once the tumor volume increased to about 1 cm3, six groups of MCF7 bearing mice (n = 10 in each group) were injected with PBS (0.1 ml, caudal vein) and adriamycin (0.1 ml, 10 mg/kg), respectively. When the tumor reached 1.5 cm in any direction (defined as event-free survival analysis), 10 mice in each group were selected to measure the tumor size and weight on the 12th day after adriamycin injection.
item1584 Six-week-old, female SCID-Berge mice were purchased from Vitalriver. EC cells with IGF2BP1 overexpression or silencing or the appropriate controls (1×106) were injected into lower abdominal cavity of each mouse (n = 5 mice/group).
item1585 .
item1586 The adeno-associated viruses (AAV) that could silence METTL3 (sh-METTL3) and the negative control adeno-associated viruses (sh-NC) were obtained from WZ Biosciences Inc. (Jinan, China). APOE-/- mice were randomly divided into AS + sh-NC and AS + sh-METTL3 groups. Each group contains five mice. Mice were fed with the standard diet for 1 week to acclimatize. After 1 week of acclimation, mice were challenged with a high-fat and high-cholesterol feed H10540 (Beijing HFK BIOSCIENCE Co., Ltd., Beijing, China). The formula of the H10540 feed was shown in Supplementary File S1. After 8 weeks of HFD feeding, sh-NC or sh-METTL3 adeno-associated virus serotype 9 (AAV9, 1012 viral genome copies per mouse) were respectively delivered into mice in AS + sh-NC or AS + sh-METTL3 group through tail vein injection. At 14 weeks after HDF feeding, mice fasted overnight.
item1587 The adeno-associated viruses (AAV) that could silence METTL3 (sh-METTL3) and the negative control adeno-associated viruses (sh-NC) were obtained from WZ Biosciences Inc. (Jinan, China). APOE-/- mice were randomly divided into AS + sh-NC and AS + sh-METTL3 groups. Each group contains five mice. Mice were fed with the standard diet for 1 week to acclimatize. After 1 week of acclimation, mice were challenged with a high-fat and high-cholesterol feed H10540 (Beijing HFK BIOSCIENCE Co., Ltd., Beijing, China). The formula of the H10540 feed was shown in Supplementary File S1. After 8 weeks of HFD feeding, sh-NC or sh-METTL3 adeno-associated virus serotype 9 (AAV9, 1012 viral genome copies per mouse) were respectively delivered into mice in AS + sh-NC or AS + sh-METTL3 group through tail vein injection. At 14 weeks after HDF feeding, mice fasted overnight.
item1588 .
item1589 The adeno-associated viruses (AAV) that could silence METTL3 (sh-METTL3) and the negative control adeno-associated viruses (sh-NC) were obtained from WZ Biosciences Inc. (Jinan, China). APOE-/- mice were randomly divided into AS + sh-NC and AS + sh-METTL3 groups. Each group contains five mice. Mice were fed with the standard diet for 1 week to acclimatize. After 1 week of acclimation, mice were challenged with a high-fat and high-cholesterol feed H10540 (Beijing HFK BIOSCIENCE Co., Ltd., Beijing, China). The formula of the H10540 feed was shown in Supplementary File S1. After 8 weeks of HFD feeding, sh-NC or sh-METTL3 adeno-associated virus serotype 9 (AAV9, 1012 viral genome copies per mouse) were respectively delivered into mice in AS + sh-NC or AS + sh-METTL3 group through tail vein injection. At 14 weeks after HDF feeding, mice fasted overnight.
item1590 The adeno-associated viruses (AAV) that could silence METTL3 (sh-METTL3) and the negative control adeno-associated viruses (sh-NC) were obtained from WZ Biosciences Inc. (Jinan, China). APOE-/- mice were randomly divided into AS + sh-NC and AS + sh-METTL3 groups. Each group contains five mice. Mice were fed with the standard diet for 1 week to acclimatize. After 1 week of acclimation, mice were challenged with a high-fat and high-cholesterol feed H10540 (Beijing HFK BIOSCIENCE Co., Ltd., Beijing, China). The formula of the H10540 feed was shown in Supplementary File S1. After 8 weeks of HFD feeding, sh-NC or sh-METTL3 adeno-associated virus serotype 9 (AAV9, 1012 viral genome copies per mouse) were respectively delivered into mice in AS + sh-NC or AS + sh-METTL3 group through tail vein injection. At 14 weeks after HDF feeding, mice fasted overnight.
item1591 1 × 106 cells in 100 uL PBS (shMETTL3-1 or shNC) were respectively injected into each mouse through the tail vein. Pulmonary metastases were monitored after fourteen days using the imaging system (IVIS) Spectrum (PerkinElmer, USA).
item1592 After anesthesia (50 mg/kg pentobarbital sodium, intraperitoneal injection), the left thorax was cut to expose the heart, and the left anterior descending (LAD) coronary artery was ligated by 7/0 sterile suture. Myocardial ischemia was induced by 30 min of LAD coronary artery ligation and following 2 h of reperfusion. Sham group mice underwent the same surgical procedure without LAD coronary artery ligation.
item1593 To construct the subcutaneous tumorigenesis model, the cells were suspended in 100 uL of PBS and Matrigel matrix (BD Biosciences, USA) (1:1) and injected into the right flanks of 6-week-old female BALB/c nude mice.To construct the lymph node metastasis model, we injected 1 × 105/50 uL stably infected SCC9 cells into the left hind footpads of BALB/c mice.
item1594 To construct the subcutaneous tumorigenesis model, the cells were suspended in 100 uL of PBS and Matrigel matrix (BD Biosciences, USA) (1:1) and injected into the right flanks of 6-week-old female BALB/c nude mice.To construct the lymph node metastasis model, we injected 1 × 105/50 uL stably infected SCC9 cells into the left hind footpads of BALB/c mice.
item1595 Ten four-week-old BALB/c nude mice were injected with LINC00958-overexpressing or vector-transfected cells. Briefly, 5 × 106 cells were subcutaneously injected in the flank of mice. Four weeks after injection, the mice were sacrificed and examined by weighting.
item1596 The right flanks of mice were injected subcutaneously with 2 × 106 MiaPaCa-2 cells stably expressing shFTO and a scrambled shRNA in 100 uL PBS. Tumors were measured using an external caliper once per week, and tumor volume was calculated with the formula: (length × width2)/2.
item1597 The right flanks of mice were injected subcutaneously with 2 × 106 MiaPaCa-2 cells stably expressing shFTO and a scrambled shRNA in 100 uL PBS. Tumors were measured using an external caliper once per week, and tumor volume was calculated with the formula: (length × width2)/2.
item1598 A tumor-bearing model was established by subcutaneously injecting 100 ul HT29 cells (5×106) followed by an intravenous injection of CAFs-derived exosomes (50 ug/mouse every three days) into the tail vein of the mice. An intraperitoneal injection of 5-FU (50 mg/kg, every week) was administered on day 12.
item1599 .
item1600 A tumor-bearing model was established by subcutaneously injecting 100 ul HT29 cells (5×106) followed by an intravenous injection of CAFs-derived exosomes (50 ug/mouse every three days) into the tail vein of the mice. An intraperitoneal injection of 5-FU (50 mg/kg, every week) was administered on day 12.
item1601 A total of 1 × 106 PC3 cells or DU145 cells suspended in a mixture of 100 uL PBS and Matrigel were subcutaneously injected into BALB/c nude mice. Tumor weight were measured 2 months after the engraftment. To evaluate the role of METTL3 in tumor metastasis, PC3 cells with or without knockdown of METTL3 were injected into SCID mice through the tail vein (1 × 106 cells per mouse). After eight weeks, mice were sacrificed and their lung tissues were collected for subsequent analyses.
item1602 A total of 1 × 106 PC3 cells or DU145 cells suspended in a mixture of 100 uL PBS and Matrigel were subcutaneously injected into BALB/c nude mice. Tumor weight were measured 2 months after the engraftment. To evaluate the role of METTL3 in tumor metastasis, PC3 cells with or without knockdown of METTL3 were injected into SCID mice through the tail vein (1 × 106 cells per mouse). After eight weeks, mice were sacrificed and their lung tissues were collected for subsequent analyses.
item1603 A total of 1 × 106 PC3 cells or DU145 cells suspended in a mixture of 100 uL PBS and Matrigel were subcutaneously injected into BALB/c nude mice. Tumor weight were measured 2 months after the engraftment. To evaluate the role of METTL3 in tumor metastasis, PC3 cells with or without knockdown of METTL3 were injected into SCID mice through the tail vein (1 × 106 cells per mouse). After eight weeks, mice were sacrificed and their lung tissues were collected for subsequent analyses.
item1604 A total of 1 × 106 PC3 cells or DU145 cells suspended in a mixture of 100 uL PBS and Matrigel were subcutaneously injected into BALB/c nude mice. Tumor weight were measured 2 months after the engraftment. To evaluate the role of METTL3 in tumor metastasis, PC3 cells with or without knockdown of METTL3 were injected into SCID mice through the tail vein (1 × 106 cells per mouse). After eight weeks, mice were sacrificed and their lung tissues were collected for subsequent analyses.
item1605 After the animal was anesthetized with isoflurane, a midline incision in the lower lumbar back region was made and the lumbar articular process was exposed and then removed. The exposed DRG was injected with viral solution (1-1.5 ul in rats and 0.5-1 ul in mice) through a glass micropipette connected to a Hamilton syringe. The pipette was retained for 10 min after injection. Animals showing signs of paresis or other abnormalities were excluded. The injected DRGs were stained with hematoxylin/eosin to examine the integrity of their structure and whether they contained visible leukocytes.
item1606 .
item1607 HCT-116 cells (3 × 105 cells in 200 uL of saline) were subcutaneously injected into the nude mice to establish xenograft tumors. After 10 days, 10 mg/kg OX or saline was intraperitoneally injected (n = 5 for each group). Si-METTL3 or si-TRAF5 (10 nmol/20 g body weight) was injected twice intratumorally before the start of OX treatment. The mice were examined every 2 days and sacrificed 4 weeks after the OX treatment.
item1608 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1609 OP rats were fixed prone, the skin was prepared and the skull was disinfected, resulting in a full-thickness defect of 8mm. BCP incubated with OP-BMSCs was implanted in the skull defect area. After 8 weeks, skull specimens were taken after the rats were euthanized.
item1610 For the mouse model of miR-320a-3p overexpression, a total of 24 male C57BL/6 mice were divided randomly into four groups (n = 6 in each group): saline, silica, silica plus AAV9-miR-NC, and silica plus AAV9-miR-320a-3p. The mice in the silica plus AAV9-miR-NC/AAV9-miR-320a-3p groups were anesthetized using the same method, then administered intratracheally 50 uL AAV9-miR-NC/AAV9-miR-320a-3p per mouse at a titer of 8 × 1012 v. g./ml. Three weeks later, these mice were treated in the same way using anesthesia, saline, and silica as mentioned above. Subsequently, after 4 weeks, the mice were sacrificed, and the lungs were isolated and frozen at -80 ℃ for further study.
item1611 For the mouse model of miR-320a-3p overexpression, a total of 24 male C57BL/6 mice were divided randomly into four groups (n = 6 in each group): saline, silica, silica plus AAV9-miR-NC, and silica plus AAV9-miR-320a-3p. The mice in the silica plus AAV9-miR-NC/AAV9-miR-320a-3p groups were anesthetized using the same method, then administered intratracheally 50 uL AAV9-miR-NC/AAV9-miR-320a-3p per mouse at a titer of 8 × 1012 v. g./ml. Three weeks later, these mice were treated in the same way using anesthesia, saline, and silica as mentioned above. Subsequently, after 4 weeks, the mice were sacrificed, and the lungs were isolated and frozen at -80 ℃ for further study.
item1612 .
item1613 ICC tumor cells (LIPF178c-shCtrl/shALKBH5) of 5 × 106 were injected into the right flank of NCG mice. Tumor volume was calculated by the formula: volume = ab2/2 (a, the longer axis; b, the shorter axis). T-cell killing assay in vitro was conducted as previously reported (20). PBMCs from healthy donors were activated and expanded as described above. The day before tumor cell injection, PBMC (i.v. 1 × 107 cells) was adoptively transferred to NCG mice via the tail vein. At the end, the PBMC was isolated and subjected to flow cytometry for detecting T-cell percentage.
item1614 .
item1615 A549 cells (1 × 106 cells) were intraperitoneally injected into the abdomen of nude mice. Tumor length and width were measured and recorded every 4 days after inoculation. Tumor volume was calculated as 1/2 × length × width2. The tumor weight was weighed and recorded 20 days after inoculation.
item1616 Before the tests, animals were fasted for 8 h. l glucose tolerance test (GTT) was conducted during week 11 on the diet. The mice were challenged with 2 g/kg body weight d-glucose (Sigma-Aldrich, USA). Insulin tolerance test (ITT) was conducted during week 12 on the diet. For insulin tolerance test, mice were injected intraperitoneally with 0.75 U/kg body weight insulin (Sigma-Aldrich, USA). For both tests, blood samples were taken from the tail vein and glucose levels were measured at indicated time points after administration using an AlphaTRAK glucometer
item1617 Before the tests, animals were fasted for 8 h. l glucose tolerance test (GTT) was conducted during week 11 on the diet. The mice were challenged with 2 g/kg body weight d-glucose (Sigma-Aldrich, USA). Insulin tolerance test (ITT) was conducted during week 12 on the diet. For insulin tolerance test, mice were injected intraperitoneally with 0.75 U/kg body weight insulin (Sigma-Aldrich, USA). For both tests, blood samples were taken from the tail vein and glucose levels were measured at indicated time points after administration using an AlphaTRAK glucometer
item1618 H1299 cells were transfected with sh-ABHD11-AS1 and harvested from six-well plates, then resuspended at a density 1 × 107 cells/ml. Mice were subsequently injected with 100 uL suspension at the right flank. After injection, tumor weight and length were examined every 3 days.
item1619 Mice were randomized into three groups (n = 7/group), 1 × 107 PC9 cells absorbed exosomes were subcutaneously injected into the Bilateral groin of mice. Treatment began 1 week following injection, the mice were intraperitoneally injected with gefitinib (30 mg/kg/day).
item1620 .
item1621 Mice were randomized into three groups (n = 7/group), 1 × 107 PC9 cells absorbed exosomes were subcutaneously injected into the Bilateral groin of mice. Treatment began 1 week following injection, the mice were intraperitoneally injected with gefitinib (30 mg/kg/day).
item1622 .
item1623 To construct the metastasis model, 5 × 106 FaDu cells were transfected with sh-IGF2BP2-luc and sh-NC-luc, suspended in 60 uL PBS, and then injected into the footpads of the mice. Six weeks after injection, mice were subjected to bioluminescence imaging to evaluate lymphatic metastasis. For bioluminescence imaging, mice were anesthetized by inhaling 2% isoflurane for approximately 5 min, injected intraperitoneally with D-Luciferin potassium salt (200 uL, 150 ug/ml, ST196, Beyotime, Shanghai, China), and imaged with a bioluminescence system (NightOwl II LB983, Berthold Technologies, Germany).
item1624 Approximately 5 × 106 control and ILF3-AS1 silencing Huh7 cells were subcutaneously implanted into the right flank of nude mice.Xenograft size was measured every 7 days and calculated using the equation V(mm3)=(length×width2)/2. 35 days later, the mice were sacrificed, and the tumor tissues were isolated and weighed.
item1625 Approximately 5 × 106 control and ILF3-AS1 silencing Huh7 cells were subcutaneously implanted into the right flank of nude mice.Xenograft size was measured every 7 days and calculated using the equation V(mm3)=(length×width2)/2. 35 days later, the mice were sacrificed, and the tumor tissues were isolated and weighed.
item1626 .
item1627 For the tumor growth analysis, AGS cells were subcutaneously injected into nude mice, and then the tumor volumes were monitored every 5 days. Tumor volumes were estimated based on the length and width and calculated using the following formula: tumor volume = (length × width2)/2. About 1 month later, the nude mice were sacrificed, and then tumors were excised, pictured, and weighed. For the tumor metastasis analysis, AGS cells were injected into nude mice by Tail Vein. About 1 month later, the nude mice were sacrificed, and then lung with metastasis lesions were excised, pictured, and counted.
item1628 .
item1629 For the xenograft model, METTL3 stable overexpressed SW620 cells (1 × 107) or control cells were subcutaneously injected into the right axilla of the female anesthetized BALB/C nude mice (4-6 weeks old, 18-20 g, four mice per group), respectively. The body weight and tumor volumes (length × width2 × 0.5) were measured twice a week. After 21 days, all mice were sacrificed and tumors were surgically removed for hematoxylin-eosin (H&E) staining.For the metastasis model, MTTL3 stable overexpressed SW620 cells (1 × 106) or control cells were injected into the exposed spleen of the anesthetized BALB/C nude mice, respectively. After 21 days, liver metastases were carefully detected using a fluorescent stereoscope and embedded for H&E staining.
item1630 For the xenograft model, METTL3 stable overexpressed SW620 cells (1 × 107) or control cells were subcutaneously injected into the right axilla of the female anesthetized BALB/C nude mice (4-6 weeks old, 18-20 g, four mice per group), respectively. The body weight and tumor volumes (length × width2 × 0.5) were measured twice a week. After 21 days, all mice were sacrificed and tumors were surgically removed for hematoxylin-eosin (H&E) staining.For the metastasis model, MTTL3 stable overexpressed SW620 cells (1 × 106) or control cells were injected into the exposed spleen of the anesthetized BALB/C nude mice, respectively. After 21 days, liver metastases were carefully detected using a fluorescent stereoscope and embedded for H&E staining.
item1631 .
item1632 .
item1633 .
item1634 .
item1635 HL-60 cells (1 × 107) suspended in 0.1 ml PBS containing 50% Matrigel were subcutaneously injected into the flanks of the mice. When tumor sizes reached 200 mm3, the mice were randomly distributed into four groups with the indicated dosages of saline, cytarabine and BP alone or in combination. For BP injections, the solution was delivered intraperitoneally at 106 ug/kg body weight for the first 8 consecutive days. For cytarabine injections, the solution was delivered intraperitoneally at 100 mg/kg body weight three times (once every three days). The combination group was administered intraperitoneally three times (once every three days) with the same dosages as described above. The control group was treated with an equivalent amount of saline.
item1636 HL-60 cells (1 × 107) suspended in 0.1 ml PBS containing 50% Matrigel were subcutaneously injected into the flanks of the mice. When tumor sizes reached 200 mm3, the mice were randomly distributed into four groups with the indicated dosages of saline, cytarabine and BP alone or in combination. For BP injections, the solution was delivered intraperitoneally at 106 ug/kg body weight for the first 8 consecutive days. For cytarabine injections, the solution was delivered intraperitoneally at 100 mg/kg body weight three times (once every three days). The combination group was administered intraperitoneally three times (once every three days) with the same dosages as described above. The control group was treated with an equivalent amount of saline.
item1637 Mettl14f/f mice were generated as previously described with CRISPR-Cas9 technology by insertion of two loxp sites into Mettl14 genome loci. Mettl14f/f mice without Villin-Cre were used as WT controls (Mettl14 WT) for Mettl14 KO mice. Mettl14f/f mice were crossed with Lgr5-eGFP-IRES-creERT2 (Lgr5-Cre) mice to generate Mettl14 depletion in Lgr5+ stem cells.
item1638 For the lung metastasis model, stably transfected HepG2 cells (1 × 106/0.1 mL DMEM) were injected into each nude mouse through the tail vein. Five weeks later, mice were euthanized, and the lung tissues were collected.
item1639 For the lung metastasis model, stably transfected HepG2 cells (1 × 106/0.1 mL DMEM) were injected into each nude mouse through the tail vein. Five weeks later, mice were euthanized, and the lung tissues were collected.
item1640 For the lung metastasis model, stably transfected HepG2 cells (1 × 106/0.1 mL DMEM) were injected into each nude mouse through the tail vein. Five weeks later, mice were euthanized, and the lung tissues were collected.
item1641 Six-week-old nude mice were randomly divided into two groups (three mice per group) and cultured with continuous access to sterile food and water in pathogen-free sterile conditions. To establish the OSCC xenograft model, we subcutaneously injected 5 × 106 SCC-9 cells stably transfected with METTL3 shRNA or sh-NC vectors into nude mice.
item1642 .
item1643 Six-week-old nude mice were randomly divided into two groups (three mice per group) and cultured with continuous access to sterile food and water in pathogen-free sterile conditions. To establish the OSCC xenograft model, we subcutaneously injected 5 × 106 SCC-9 cells stably transfected with METTL3 shRNA or sh-NC vectors into nude mice.
item1644 In vivo, the group of HCT116 cells labeled with luciferase were surgically injected into the spleen of nude mice. After 2 months, the nude mice were subjected to D-luciferin injection and the luciferase signals were monitored and quantified.
item1645 .
item1646 .
item1647 Mice were anesthetized with Nembutal. The lower back was dissected until the transverse lumbar process was exposed. After the process was removed, the underneath L4 spinal nerve was ligated with a silk 6-0 thread. A slight distal location was chosen for transection around the ligation site. Subsequent layers of muscle and skin were closed. The sham groups undertook identical procedures, but without the transection or ligature of the corresponding nerve. The intraspinal injection was performed as described previously. In short, after anesthetized with Nembutal, mice underwent hemilaminectomy at the L1-L2 vertebral segments. The intraspinal injection was carried out ipsilaterally on the left side. By using a glass micropipette, each animal received two injections (5 × 105 TU per injection, 0.8 mm from the midline, 0.5 mm apart in rostrocaudal axis, 0.5 mm deep) of lentivirus following the L3-L4 dorsal root entry zone after exposure of spinal cord. The tip of glass micropipette should reach a depth of lamina II-IV of the spinal cord. Finally, the dorsal muscle and skin were sutured layer by layer.
item1648 U2OS cells with stable METTL3 expression were screened using puromycin for subsequent experiments. U2OS cells (1 × 106) were injected subcutaneously to the right side of each mouse (the mice were numbered according to their weight, and the experimenters randomly divided the nude mice into 12 groups by the random number method, with 12 mice in each group). The status of mice was detected every 2 days. Tumor growth and volume were measured once a week.Tumor volume was measured: Volume = Width2 × Length/2. On the 35th day after the operation, nude mice were euthanized by intraperitoneal injection of ≥100 mg/kg pentobarbital sodium. Tumor resection was performed and tumor weight was recorded. Weight loss >10% of the body weight or the maximum diameter of the tumor >1.5 cm was the humane end point.
item1649 .
item1650 .
item1651 .
item1652 .
item1653 .
item1654 For the tumorigenicity studies, 3 × 106 HCC827 cells stably expressing sh-LINC01833 or sh-NC were injected subcutaneously into the ventral side of male BALB/c nude mice in the according groups with five mice in each group. Five mice were sampled each time. Tumor size and weight were examined at 28 days after injection.
item1655 The model mice were intraperitoneally injected with streptozotocin (STZ; Sigma-Aldrich Corp., USA). The dose of STZ was 50 mg/kg for 5 days. 7 days after the last injection, blood glucose concentrations were recorded. Mouse models of diabetes were considered established when fasting blood glucose concentrations reached >11.1 mmol/L, and body weight was measured.
item1656 .
item1657 Male nu/nu mice between 4 and 6 weeks of age received subcutaneous injections of equivalent Hep3B cells expressing either LV-shMETTL3 or LV-USP7 within 30 min of harvesting on the right and left flanks. The tumor was weighed after approximately 4 weeks, and the volume was measured every 5 days.
item1658 For the proliferation assays, C4-2B cells of KIF3C knockdown, negative control (1×106/200uL) were subcutaneously injected into BALB/c nude mice. The tumors were dissected and weighed (4-6 weeks old, male).
item1659 .
item1660 Xenograft mouse model was used to verify the tumorigenic effect of METTL16 in vivo. BALB/c nude mice (4 weeks old) were injected with METTL16 gene knock-down stable MGC803 GC cells (3 × 106 cells/mice, subcutaneous injection) or shNC control cells (3 × 106, subcutaneous injection), and the dose was 100 uL, with PBS as solvent. The tumour size was measured every 3-5 days. At the end of feeding (6 weeks after subcutaneous injection), the mice were killed and the tumours were extracted for histological analysis.
item1661 .
item1662 C57BL/6 mouse hearts were subjected to ischemia/reperfusion (I/R) in vivo as described previously (Bock-Marquette et al., 2004; Song et al., 2015; Brocard et al., 2017). I/R injury in mice was induced by 45-min ischemia, followed by 7-day and 4-week reperfusion in a loss-of-function study (Figure 1) and gain-of-function study (Figure 2), respectively. In brief, mice were anesthetized with 2% avertin (0.1 ml/10g body weight; Sigma-Aldrich Corporation, United States) through intraperitoneal injection. To generate I/R injury, the left anterior descending coronary artery (LAD) was ligated with 7-0 nylon for 45 min and then was removed. For the sham group, a suture was passed under the LAD but without ligation. According to the experimental requirements, at different time points of cardiac I/R, the mice were anesthetized for assessing heart function by echocardiographic measurement. All the mice survived during the process of I/R injury after the operation.
item1663 Suspension of H1299 cells (5.0 × 105) was subcutaneously injected into the right flanks of the mice.
item1664 C57BL/6 mouse hearts were subjected to ischemia/reperfusion (I/R) in vivo as described previously (Bock-Marquette et al., 2004; Song et al., 2015; Brocard et al., 2017). I/R injury in mice was induced by 45-min ischemia, followed by 7-day and 4-week reperfusion in a loss-of-function study (Figure 1) and gain-of-function study (Figure 2), respectively. In brief, mice were anesthetized with 2% avertin (0.1 ml/10g body weight; Sigma-Aldrich Corporation, United States) through intraperitoneal injection. To generate I/R injury, the left anterior descending coronary artery (LAD) was ligated with 7-0 nylon for 45 min and then was removed. For the sham group, a suture was passed under the LAD but without ligation. According to the experimental requirements, at different time points of cardiac I/R, the mice were anesthetized for assessing heart function by echocardiographic measurement. All the mice survived during the process of I/R injury after the operation.
item1665 All the mice (n = 12) were equally and randomly divided into the HCT-scr and HCT-shMETTL3 group. 3 × 106 HCT-scr or HCT-shIGF2BP3 cells suspended in 100 uL PBS were injected subcutaneously from the axilla of each nude mice. After 1 weeks, the long (L) and short (S) diameter of the tumors were measured with vernier caliper every 3 days (tumor volume = L*S2/2). The growth curve of subcutaneous tumors was drawn on the basis of the measured tumor volume. All mice were killed after 17 days since injection of colon cancer cells and subcutaneous tumors were removed completely.
item1666 For the subcutaneous transplantation model, sh-control, sh-FTO, NC-OE and ERBB2-OE KYSE150 stable cells (6 × 106 per mouse, n = 3 for each group) were diluted to 100 uL PBS + 100 uL Matrigel (BD) and subcutaneously injected to two points in the middle and upper groin of immunodeficient mice to study tumor growth. When the tumor volume reached ~ 1000 mm3 in each group, the nude mice were killed, and the tumors were excised and weighed for histology and further study. The tumor volume was calculated by the formula V = 1/2 × large diameter × (small diameter)2. For a model of lung metastasis in vivo, nude mice were injected with 100 uL WT (wide type), sh-FTO, ERBB2-OE and sh-FTO+ERBB2-OE KYSE150 stable cells (1 × 106 cells per mouse, n = 3 for each group) through tail vein, respectively. Six weeks after injection, the mice were killed and analyzed for metastatic lung tumors.
item1667 .
item1668 .
item1669 .
item1670 Female C57BL/6J mice (14 weeks old) were treated with bilateral ovariectomy under general anesthesia. Eight weeks after surgery, tibial plateau was harvested and the structure were evaluated with a SCANCO Medical uCT 40 scanner.
item1671 .
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item1673 .
item1674 Mice were subcutaneously injected with 2.5 × 106 U251 cells stably infected with OE-NC, OE-JMJD1C, OE-NC and sh-NC, OE-JMJD1C and sh-NC or OE-JMJD1C and sh-SOCS2 (n = 10 in each group) in 0.1 ml PBS.
item1675 For the purpose of enhancing the overall randomization of the experiment, a random comparison table had been employed. Accordingly, 5-wk-old male nude athymic BALB/c nu/nu mice (Slack, Shanghai, China) were randomly divided into two parts including a control group (NC) and the experimental group METTL14-OE. For developing subcutaneous xeno transplantation model, 5 × 106 HGC-27 cells stably transfected with NC or METTL14 overexpression were subcutaneously incorporated for 5-week-old BALB/c nude mice. The mice experienced euthanasia after 27 days of inoculation and obtained xenografts's mass was obtained. Tumor volume over three days was obtained. To create mouse STAD liver metastasis orthotopic tumor model, 1 × 106 HGC-27 cells under stable transfection with NC or METTL14 overexpression were added to subserosal gastric wall of BALB/c nude mice.
item1676 BALB/c-nu mice were grouped as follows to detect tumor growth: HeLa-sh-NC [mice inoculated with scramble shRNA-infected control HeLa cells (n = 5)] and HeLa-sh-CENPK [mice inoculated with CENPK-targeted shRNA-infected HeLa cells (n = 5)]. The mice were subcutaneously inoculated with 5 × 106 cells/100 uL in the flank. The longest and the shortest diameters of the growing tumors were measured every 3 d with a caliper, and the tumor volume (V) was counted by the following equation: V = (the longest diameter × the shortest diameter2)/2. The mice were grouped as follows to evaluate the tumor-initiating frequency and were inoculated with a series of 5 × 105, 2 × 105, and 5 × 104 cells subcutaneously: HeLa-sh-NC (n = 6); and HeLa-sh-CENPK (n = 6). The mice bearing subcutaneous xenograft tumors were grouped as follows to evaluate tumor chemoresistance: HeLa-sh-NC (n?=?10); HeLa-sh-CENPK (n?=?10); HeLa-sh-NC?+?cisplatin [mice inoculated with scramble shRNA-infected control HeLa cells and 3 mg/kg of cisplatin once a week intraperitoneally (ip) for 6 weeks (n = 10)]
item1677 Stable H1299 cell lines diluted into 100uL were injected subcutaneously into 5-week-old nude mice (n = 5) at the final concentration of 3 × 106 cells. Mice were sacrificed 4 weeks later, and tumors were weighed and photographed.
item1678 For xenograft models, 5 × 106 BCPAP or KTC-1 cells from each group were injected subcutaneously into the flanks of female BALB/c nude mice (4-6 weeks old, Shanghai SLAC Laboratory Animal, China, n = 5 per group) in a volume of 150 uL PBS. Tumor growth was measured with a digital caliper every 4 days and calculated using the following formula: (length × width2)/2. To study the effect of IL-8 on tumor growth in vivo, scramble or shMETTL3 BCPAP cells were implanted hypodermically into BALB/c nude mice (2 × 106 cells in 150 uL PBS, n = 10 per group). When palpable tumors formed on day 14, mice were treated with DMSO or the IL-8 inhibitor SB225002 (10 mg/kg) by intraperitoneal injection 3 times per week for 3 weeks. Six weeks post-injection, the mice were sacrificed, and the tumors were collected to analyze the frequency of TANs by flow cytometry. For the lung metastasis model, BCPAP and KTC-1 cells (2 × 106 cells in 100 uL PBS) with the corresponding vectors were injected into the tail veins of BALB/c nude mice. Eight weeks after injection, the mice were euthanized, and metastatic lung nodules were analyzed (n = 5 for each group).
item1679 For xenograft models, 5 × 106 BCPAP or KTC-1 cells from each group were injected subcutaneously into the flanks of female BALB/c nude mice (4-6 weeks old, Shanghai SLAC Laboratory Animal, China, n = 5 per group) in a volume of 150 uL PBS. Tumor growth was measured with a digital caliper every 4 days and calculated using the following formula: (length × width2)/2. To study the effect of IL-8 on tumor growth in vivo, scramble or shMETTL3 BCPAP cells were implanted hypodermically into BALB/c nude mice (2 × 106 cells in 150 uL PBS, n = 10 per group). When palpable tumors formed on day 14, mice were treated with DMSO or the IL-8 inhibitor SB225002 (10 mg/kg) by intraperitoneal injection 3 times per week for 3 weeks. Six weeks post-injection, the mice were sacrificed, and the tumors were collected to analyze the frequency of TANs by flow cytometry. For the lung metastasis model, BCPAP and KTC-1 cells (2 × 106 cells in 100 uL PBS) with the corresponding vectors were injected into the tail veins of BALB/c nude mice. Eight weeks after injection, the mice were euthanized, and metastatic lung nodules were analyzed (n = 5 for each group).
item1680 For xenograft models, 5 × 106 BCPAP or KTC-1 cells from each group were injected subcutaneously into the flanks of female BALB/c nude mice (4-6 weeks old, Shanghai SLAC Laboratory Animal, China, n = 5 per group) in a volume of 150 uL PBS. Tumor growth was measured with a digital caliper every 4 days and calculated using the following formula: (length × width2)/2. To study the effect of IL-8 on tumor growth in vivo, scramble or shMETTL3 BCPAP cells were implanted hypodermically into BALB/c nude mice (2 × 106 cells in 150 uL PBS, n = 10 per group). When palpable tumors formed on day 14, mice were treated with DMSO or the IL-8 inhibitor SB225002 (10 mg/kg) by intraperitoneal injection 3 times per week for 3 weeks. Six weeks post-injection, the mice were sacrificed, and the tumors were collected to analyze the frequency of TANs by flow cytometry. For the lung metastasis model, BCPAP and KTC-1 cells (2 × 106 cells in 100 uL PBS) with the corresponding vectors were injected into the tail veins of BALB/c nude mice. Eight weeks after injection, the mice were euthanized, and metastatic lung nodules were analyzed (n = 5 for each group).
item1681 For xenograft models, 5 × 106 BCPAP or KTC-1 cells from each group were injected subcutaneously into the flanks of female BALB/c nude mice (4-6 weeks old, Shanghai SLAC Laboratory Animal, China, n = 5 per group) in a volume of 150 uL PBS. Tumor growth was measured with a digital caliper every 4 days and calculated using the following formula: (length × width2)/2. To study the effect of IL-8 on tumor growth in vivo, scramble or shMETTL3 BCPAP cells were implanted hypodermically into BALB/c nude mice (2 × 106 cells in 150 uL PBS, n = 10 per group). When palpable tumors formed on day 14, mice were treated with DMSO or the IL-8 inhibitor SB225002 (10 mg/kg) by intraperitoneal injection 3 times per week for 3 weeks. Six weeks post-injection, the mice were sacrificed, and the tumors were collected to analyze the frequency of TANs by flow cytometry. For the lung metastasis model, BCPAP and KTC-1 cells (2 × 106 cells in 100 uL PBS) with the corresponding vectors were injected into the tail veins of BALB/c nude mice. Eight weeks after injection, the mice were euthanized, and metastatic lung nodules were analyzed (n = 5 for each group).
item1682 .
item1683 .
item1684 PCa cells carrying the transfected plasmid were subcutaneously injected into immunodeficient mice at a rate of 1 × 106 cells per mouse according to a previous study. Tumor formation in the two groups of mice was observed and recorded by a designated personnel every day, and the volume of the tumors was measured. The nude mice were sacrificed 21 days after tumor formation, and the tumors were removed to measure their volume and weight.
item1685 .
item1686 For the mouse lung metastasis model, SUM-1315 cells (2 × 106/0.2 mL) expressing NC, shKIAA1429, SNAIL, or shKIAA1429+SNAIL were injected into the nude mice through the tail vein.
item1687 For the mouse lung metastasis model, SUM-1315 cells (2 × 106/0.2 mL) expressing NC, shKIAA1429, SNAIL, or shKIAA1429+SNAIL were injected into the nude mice through the tail vein.
item1688 The GC cell line MKN-45 stably infected with lentivirus expressing sh-HBXIP was prepared into 5 × 107 cells/mL cell suspension. The cell suspension was injected into the left axilla of nude mice using a 1 mL syringe as the sh-HBXIP group (n = 6). The GC cell line MKN-45 infected with the lentivirus expressing sh-NC was dispersed into the cell suspension, which was injected into nude mice as the sh-NC group (n = 6). Tumor growth was observed and data were recorded after inoculation. On the 26th day, all nude mice were euthanized by cervical dislocation and the tumors were resected and weighed.
item1689 .
item1690 Female SCID-Beige mice (6 weeks old) were purchased from Vitalriver. AN3CA cells with FTO overexpression or silencing or the appropriate controls (1 × 106) were injected into the lower abdominal cavity of mice (n = 5 mice/group). After 4 weeks, the mice were sacrificed, and tumours were harvested, weighed and photographed.
item1691 The diabetic model was constructed by a single intraperitoneal injection of streptozotocin (65 mg/kg), which imitates a model of type 1 diabetes. The fasting blood glucose was measured one week after injection. Only rats with glucose levels higher than 16.7 mmol/L were defined as diabetic. Cardiac function was investigated seven days following the last treatment, and the heart tissues were then isolated for expression analyses. The lentivirus vector used for silencing or overexpressing specific genes were dissolved in 50uL saline at the concentration of 1 × 109 TU with one dose after the animal model was established. NLRP3 inhibitor MCC950 (10 mg/kg) was intraperitoneally injected 30 min before streptozotocin treatment.
item1692 About 5× 106 cells were injected subcutaneously into the axilla of the female athymic BALB/C nude mice (4 week-old, 18-20?g). When the average tumor size reached approximately 100mm3 (after 1 week), mice were then randomized into two groups and treated with cisplatin (5 mg/kg) or normal saline (NS) weekly.
item1693 4-week-old female BALB/c nude mice were used for HuCC-T1 tumor xenograft models and 4-week-old female B-NDG mice (Biocytogen, Beijing, China) were used for HCCC-9810 tumor xenograft models. 1 × 107 HuCC-T1 or HCCC-9810 cells were resuspended in 100 ul PBS with Matrigel (1:1), and injected into the right flank of mice (n = 6/group).
item1694 Lentiviruses containing sh-METTL-14 and its negative control (RiboBio Co., Ltd., Guangzhou, China) were transduced into CAL27 cells and stably transduced cells were screened using puromycin. CAL27 cells (3 × 106 cells/mouse) were subcutaneously inoculated into the posterior flank of each mouse (N = 12/group).
item1695 BCPAP cells (5×106) were introduced into the mice by means of subcutaneous injection through the flank area. STEAP2-saRNA or NC-saRNA (n = 6 for each group) was given by intratumoral multipoint injection at an interval of 3 days (5 injections in total) using an in vivo transfection reagent (Entranster -in vivo, Engreen, China) as per the vendor-provided protocol. Tumor volume (V) was monitored and calculated as follows: V = (L×W2)/2. For the in vivo tumor metastasis assay, BCPAP cells (5×106 cells) were administrated into mice through the tail vein. STEAP2-saRNA or NC-saRNA (n = 6 for each group) was given via tail vein injection at an interval of 3 days (8 injections in total).
item1696 .
item1697 .
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item1699 .
item1700 .
item1701 Male BALB/c-nu/nu mice, 9-10 weeks of age, were acclimatized for a week prior to the experiments. Nude mice (6 each group) were subcutaneously inoculated with 4 × 106 SCC-25 or SCC-15 cells through an injection into the center of the back, which consistently caused tumor formation within 1 week of inoculation. To monitor the initial tumor appearance, animals were observed every day. After tumor appeared, measurements were made every week with a caliper. After 28 days, mice were sacrificed and tumors were dissected out to be weighed.
item1702 Ten four-week-old BALB/c male nude mice (GemPharmatech, Jiangsu, China) were subcutaneously injected with control Huh7 cells 2 × 106 (left-back) and stable knockdown of YTHDF1 Huh7 cells 2 × 106 (right-back). These cells were respectively premixed with 50 ul Matrigel (Corning, 354,234) in 100 ul PBS.
item1703 Ten four-week-old BALB/c male nude mice (GemPharmatech, Jiangsu, China) were subcutaneously injected with control Huh7 cells 2 × 106 (left-back) and stable knockdown of YTHDF1 Huh7 cells 2 × 106 (right-back). These cells were respectively premixed with 50 ul Matrigel (Corning, 354,234) in 100 ul PBS.
item1704 For the subcutaneously transplanted tumor model, wild-type, PRMT5-overexpressing or doxorubicin-resistant MDA-MB-231 cells (5 × 106 per mouse, n = 5-7 for each group) were diluted in 100 uL of phosphate-buffered saline (PBS) plus 100 uL of Matrigel (BD Biosciences) and subcutaneously injected into female nude mice to investigate tumor growth. When all tumor volumes reached 100 mm3, the mice were randomly assigned and treated with the indicated drugs. In the experiment, doxorubicin was administered once a week via intravenous tail vein injection at 2 mg/kg body weight, and tadalafil was administered daily via oral gavage at 2 mg/kg body weight. Tumor volume was measured every 3 days using a digital caliper and calculated using the formula V = 1/2 × (diameter) × (smaller diameter)2. The mice were euthanized 27 days after injection.
item1705 .
item1706 .
item1707 For subcutaneous xenotransplanted tumor models, cells were injected subcutaneously (5 × 106 for MHCC97H or 1×106 for PLC cells per mouse).
item1708 .
item1709 Tumour xenograft models were established in nude mice bearing: SAS cells cells stably transfected with METTL3-shRNA and the corresponding control vector. The different HNSCC cells (5 × 106) were subcutaneously injected into the right axilla of nude mice (n = 6 per group).
item1710 .
item1711 Cells were trypsinized and resuspended in DMEM at a consistence of 1 × 107 cells/ml. A total of 1 × 106 cells were injected into flank of mice. 27 days after injection, tumors were removed for paraffin-embedded sections.
item1712 Cells were trypsinized and resuspended in DMEM at a consistence of 1 × 107 cells/ml. A total of 1 × 106 cells were injected into flank of mice. 27 days after injection, tumors were removed for paraffin-embedded sections.
item1713 .
item1714 .
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item1717 A total of 8 × 106 wild-type (WT) or METTL3-knockdown cells were injected into the dorsal flanks of 6-week-old nude mice. Seven mice were randomly selected to calculate the volume according to the following formula: V = (width2 × length)/2. Mice were euthanized three weeks after injection and tumors removed, weighed, fixed, and embedded for immunohistochemical analysis.
item1718 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1719 To generate an AMI mouse model, mice were anesthetised by intraperitoneal injection of sterile pentobarbital sodium at 50 mg/kg body weight.
item1720 A total of 8 × 106 wild-type (WT) or METTL3-knockdown cells were injected into the dorsal flanks of 6-week-old nude mice. Seven mice were randomly selected to calculate the volume according to the following formula: V = (width2 × length)/2. Mice were euthanized three weeks after injection and tumors removed, weighed, fixed, and embedded for immunohistochemical analysis.
item1721 A total of 8 × 106 wild-type (WT) or METTL3-knockdown cells were injected into the dorsal flanks of 6-week-old nude mice. Seven mice were randomly selected to calculate the volume according to the following formula: V = (width2 × length)/2. Mice were euthanized three weeks after injection and tumors removed, weighed, fixed, and embedded for immunohistochemical analysis.
item1722 A total of 8 × 106 wild-type (WT) or METTL3-knockdown cells were injected into the dorsal flanks of 6-week-old nude mice. Seven mice were randomly selected to calculate the volume according to the following formula: V = (width2 × length)/2. Mice were euthanized three weeks after injection and tumors removed, weighed, fixed, and embedded for immunohistochemical analysis.
item1723 A total of 8 × 106 wild-type (WT) or METTL3-knockdown cells were injected into the dorsal flanks of 6-week-old nude mice. Seven mice were randomly selected to calculate the volume according to the following formula: V = (width2 × length)/2. Mice were euthanized three weeks after injection and tumors removed, weighed, fixed, and embedded for immunohistochemical analysis.
item1724 A total of 8 × 106 wild-type (WT) or METTL3-knockdown cells were injected into the dorsal flanks of 6-week-old nude mice. Seven mice were randomly selected to calculate the volume according to the following formula: V = (width2 × length)/2. Mice were euthanized three weeks after injection and tumors removed, weighed, fixed, and embedded for immunohistochemical analysis.
item1725 Stable short hairpin (shRNA)-expressing MGC-803 cells (3 × 106) were suspended in 0.1 mL PBS and injected into the flanks of BALB/c mice (n = 8 mice/group) at 5-6 weeks of age. For the flanks injected mice, tumor growth was examined every 3 days. After 5 weeks, mice were sacrificed by cervical dislocation, and weight of xenografts was tested.BALB/c mice were randomly divided into three groups (n = 4 mice/group). A total of 3 × 106 stable shRNA-expressing MGC-803 cells were resuspended in 0.1 mL PBS and injected into the abdominal cavity. After 4 weeks, mice were sacrificed by cervical dislocation, abdominal cavities were opened, and the numbers of implantation metastasis were counted.For the pulmonary metastasis model, NOD/SCID mice were randomly divided into three groups (n = 4 mice/group). A total of 1 × 105 stable shRNA-expressing MGC-803 cells were resuspended in 0.1 mL PBS and injected into the lateral tail vein. After 7 weeks, mice were sacrificed by cervical dislocation, and lungs were extracted and fixed 4% paraformaldehyde in PBS. Paraffin embedding, sectioning, and staining with hematoxylin and eosin were performed. Visible lung metastases were measured and counted using a microscope.
item1726 Specifically, rats were anesthetized with an intraperitoneal injection of 4% pentobarbital sodium (35 mg/kg) after weight measurement. To expose the posterior vertebral arch from T8 to T12, an incision was subsequently made on the skin along the dorsomedial line to the aponeurosis and muscle plane. Laminectomy (3 mm) was performed from the caudal end of the T10 vertebra to the caudal end of the T11 vertebra under a dissection stereomicroscope. Infinite Horizons impactor (Infinite Horizons, L.L.C., Lexington, KY, USA) was utilized to induce contusion SCI at the force of 60 kdyn/cm . The incision was sutured, followed by intramuscular injection of 20000 units of penicillin once a day for three days. Incisions of rats in the sham group (N = 10) were sutured after skin incision without modeling surgery and related treatment. SCI rat model was established and SCI rats were assigned to the following groups (N = 10 per group): SCI group (SCI treatment), SCI + sh-NC group (injected with silencing negative control lentivirus after SCI treatment), SCI + sh-METTL14 + sh-EEF1A2 group (injected with silencing EEF1A2 and silencing METTL14 lentivirus after SCI treatment), SCI + oe-NC group (injected with overexpressed EEF1A2 NC lentivirus after SCI treatment), SCI + oe-EEF1A2 group (injected with overexpressed EEF1A2 lentivirus after SCI treatment), SCI + oe-EEF1A2 + H2O group [injected with overexpressed EEF1A2 lentivirus and treated with 50 mg/kg (i.p.) H2O after SCI treatment] and SCI + oe-EEF1A2 + Perifosine group [injected with overexpressed EEF1A2 lentivirus and treated with 50 mg/kg (i.p.) Perifosine after SCI treatment . Lentivirus treatment was conducted three days following laminectomy (on day 0, 1, and 2). sh-NC, sh-METTL14, sh-EEF1A2, oe-NC, and oe-EEF1A2 lentivirus (50 uL/day, 100 nmoL/mL; RiboBio, Guangzhou, China) were intrathecally injected through lumbar puncture for 15 min per day.
item1727 Male SD rats weighing 200-250 g were anesthetized using 4% isoflurane in 70% N2O and 30% O2 with a mask. A midline incision was made in the neck, the left external carotid artery (ECA) was carefully exposed and dissected, a monofilament nylon suture with a diameter of about 0.22 mm was inserted from the ECA into the internal carotid artery, and the left middle cerebral artery (MCA) was blocked. After occlusion for 90 minutes, the suture was removed for reperfusion, and ECA was ligated to close the wound. Sham-operated rats underwent the same surgery except for suture insertion. Rats were maintained on top of a warming pad (RWD, 69003) during the above procedures. The breathing machine was used to monitor the respiration of rats. The rats were returned to a heated cage during the recovery phase with free access to food and water.
item1728 .
item1729 The nude mice were maintained under pathogen-free conditions and kept under timed lighting conditions mandated by the committee with food and water provided ad libitum. For xenograft experiments, nude mice were injected subcutaneously with 5 × 106 cells resuspended in 0.1 mL PBS. When a tumor was palpable, it was measured every 3 days.
item1730 .
item1731 The nude mice were maintained under pathogen-free conditions and kept under timed lighting conditions mandated by the committee with food and water provided ad libitum. For xenograft experiments, nude mice were injected subcutaneously with 5 × 106 cells resuspended in 0.1 mL PBS. When a tumor was palpable, it was measured every 3 days.
item1732 .
item1733 Nude mice were randomly divided into five groups of six mice each, and the mice in each group were received subcutaneous injections of shFTO, scrambled shNC, FTO OE, and vector KYSE510 cells (5×106 tumor cells/mouse), respectively. The tumor size and weight of mice were measured 1 week later, which was recorded as day 0, and then measured once every other day; and the tumor volumes were calculated using the following formula: (length×width2)/2.When the tumor maximum diameter was close to 15 mm, the mice were euthanized and the tumor tissues were collected for immunohistochemistry analysis.
item1734 .
item1735 .
item1736 NOD/SCID immune-deficient mice were purchased from Shanghai Experimental Animal Center. 2 * 106 MCF-7 cells transduced with sh-NC or sh-YTHDF1-were subcutaneously injected into the mice (5/group). Tumor width and length were measured every 7 days. Tumor volume?=?(length * width2)/2. After 7 weeks, mice were sacrificed, and the weight of tumors was detected. Xenografts were collected for HE staining, immunohistochemistry staining and western blot analysis.For spontaneous lung metastasis assay, 4 * 106 sh-NC or sh-YTHDF1#2 transduced MCF-7 cells were injected into the mammary fat pads of the NOD/SCID mice (5/group). The primary tumor was removed when its volume reached 150 mm3. The mice were sacrificed, and lung metastasis nodules were counted 12 weeks after the removal.
item1737 For the in vivo brain and bone extravasation and seeding assays, cancer cells labeled with CMFDA C2925 (Thermo fisher scientific) or GFP were injected intracardially into the nude mice. Cell number and injection procedure were described in "Animal Experiments". For the in vivo lung extravasation and seeding assays, cancer cells labeled with GFP (2.5 × 105 cells/mouse) were injected into the tail vein of nude mice. At 24 or 48 hrs later, the mice were sacrificed.
item1738 For the in vivo brain and bone extravasation and seeding assays, cancer cells labeled with CMFDA C2925 (Thermo fisher scientific) or GFP were injected intracardially into the nude mice. Cell number and injection procedure were described in "Animal Experiments". For the in vivo lung extravasation and seeding assays, cancer cells labeled with GFP (2.5 × 105 cells/mouse) were injected into the tail vein of nude mice. At 24 or 48 hrs later, the mice were sacrificed.
item1739 For the in vivo brain and bone extravasation and seeding assays, cancer cells labeled with CMFDA C2925 (Thermo fisher scientific) or GFP were injected intracardially into the nude mice. Cell number and injection procedure were described in "Animal Experiments". For the in vivo lung extravasation and seeding assays, cancer cells labeled with GFP (2.5 × 105 cells/mouse) were injected into the tail vein of nude mice. At 24 or 48 hrs later, the mice were sacrificed.
item1740 Approximately 2×106 PCa cells (DU145 transfected with shFTO and shNC) were injected subcutaneously in mice. The tumor volume (V = (0.5*length*width2)) was measured with Vernier caliper every week. Ten mice were randomly divided into two groups, 2×106 cells transfected with shFTO and shNC were resuspended with 100 uL PBS and injected into the mouse tail vein to create a metastatic model. After 7 weeks, the mice were anesthetized, and D-luciferin (#D-Luciferin, Apexbio) was injected intraperitoneally, then used the IVIS imaging system (Caliper Life Sciences) to visualize the luciferase signal.
item1741 To generate doxycycline-inducible skeletal muscle-specific FTO deletion mice, FTOflox/flox mice were crossed with HSA-Cre mice to generate FTOflox/+ HSA-Cre mice, which were then crossed to FTOflox/flox mice to generate FTOflox/flox and FTOflox/flox HSA-Cre mice.
item1742 .
item1743 .
item1744 .
item1745 .
item1746 Female BALB/c nude mice (ages 4-5 weeks, 18-20 g) were purchased from the Charles River Laboratories. For the tumor growth model, 1 × 106 HONE-1 sh-NC or sh-ZFAS1 cells were injected into the axilla of the mice, and the tumor size was measured every 3 days. On day 30, the mice were killed, and the tumors were dissected and weighed.
item1747 .
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item1750 .
item1751 A total of 30 BALB/c nude mice were chosen and assigned to three groups: (1) control (injected with 0.2 mL PBS), (2) si-NC (injected with si-NC transfected SGC7901 cells) and (3) si-IGF2BP2 (injected with si-IGF2BP2 transfected SGC7901 cells (n = 5 per group). 2 × 106 SGC7901 cells were injected into the left right back of each mouse through subcutaneous injection. Tumor sizes were recorded once per week. After 28 days, the mice were euthanized, and tumor tissues were weighted.
item1752 .
item1753 .
item1754 Five-week-old female BALB/c nude mice (Charles Rivers, Beijing, China) were selected for the experiments. U87 cells (5 × 105) transfected with an empty vector, YTHDF2 overexpression, or METTL3 overexpression vectors were suspended in PBS and injected into the right frontal node of nude mice. The inoculation position was 2 mm lateral and 2 mm posterior to the anterior fontanel. Tumor size was estimated from luciferase volume measurements and MRI. The mice were sacrificed when they exhibited disturbed activity or convulsion. The brain was then harvested and embedded in paraffin.
item1755 Five-week-old female BALB/c nude mice (Charles Rivers, Beijing, China) were selected for the experiments. U87 cells (5 × 105) transfected with an empty vector, YTHDF2 overexpression, or METTL3 overexpression vectors were suspended in PBS and injected into the right frontal node of nude mice. The inoculation position was 2 mm lateral and 2 mm posterior to the anterior fontanel. Tumor size was estimated from luciferase volume measurements and MRI. The mice were sacrificed when they exhibited disturbed activity or convulsion. The brain was then harvested and embedded in paraffin.
item1756 Five-week-old female BALB/c nude mice (Charles Rivers, Beijing, China) were selected for the experiments. U87 cells (5 × 105) transfected with an empty vector, YTHDF2 overexpression, or METTL3 overexpression vectors were suspended in PBS and injected into the right frontal node of nude mice. The inoculation position was 2 mm lateral and 2 mm posterior to the anterior fontanel. Tumor size was estimated from luciferase volume measurements and MRI. The mice were sacrificed when they exhibited disturbed activity or convulsion. The brain was then harvested and embedded in paraffin.
item1757 Five-week-old female BALB/c nude mice (Charles Rivers, Beijing, China) were selected for the experiments. U87 cells (5 × 105) transfected with an empty vector, YTHDF2 overexpression, or METTL3 overexpression vectors were suspended in PBS and injected into the right frontal node of nude mice. The inoculation position was 2 mm lateral and 2 mm posterior to the anterior fontanel. Tumor size was estimated from luciferase volume measurements and MRI. The mice were sacrificed when they exhibited disturbed activity or convulsion. The brain was then harvested and embedded in paraffin.
item1758 After randomly assignment and anesthetization, nude mice were injected with 5 × 106 cells suspended in 100 uL PBS into the tail vein (n = 5 per group).
item1759 After randomly assignment and anesthetization, nude mice were injected with 5 × 106 cells suspended in 100 uL PBS into the tail vein (n = 5 per group).
item1760 .
item1761 .
item1762 The mice were fed in an SPF environment (cycle of 12-h light and 12-h dark) with a free diet. All the mice were adaptively fed for 5 days before experiments and randomly divided into the following four groups: the siNC+MC group (n = 10), the METTL3 siRNA1+MC group (n = 10), siNC+M group (n = 10) and the METTL3 siRNA1+M group (n = 10). Then, the right forelimb of mice in the siNC+MC group was subcutaneously injected with 100 uL PBS containing 1 × 106 SK-Hep1 cells co-transfected with siNC and MC; the right forelimb of mice in the METTL3 siRNA1+MC group was subcutaneously injected with 100 uL PBS containing 1 × 106 SK-Hep1 cells co-transfected with METTL3 siRNA1 and MC; the right forelimb of mice in the siNC+M group was subcutaneously injected with 100 uL PBS containing 1 × 106 SK-Hep1 cells co-transfected with siNC and M; the right forelimb of mice in the METTL3 siRNA1+M group was subcutaneously injected with 100 uL PBS containing 1 × 106 SK-Hep1 cells co-transfected with METTL3 siRNA1 and M.
item1763 .
item1764 .
item1765 .
item1766 Mice (male and 6 weeks old) were subcutaneously injected with NSCLC cells (1.0*106 cells/200 uL). The mice were terminated after 4 weeks of induction, and the tumor volume and tumor weight were measured.
item1767 The mice were divided into control group and ALKBH5-overexpressing group (9 mice per group). ALKBH5-overexpressing and control A549 cells (3 × 106 cells/mouse) in 200 uL PBS were intravenously (i.v.) injected into the lateral tail vein of mice. At every 5th day post-inoculation, TGF-Beta-1 (4 ug/kg body weight) was intraperitoneally (i.p.) injected to promote tumor cell metastasis. Eight weeks later, the mice were euthanized, and then their lungs and livers were taken out and fixed in Bouin's solution (Sigma Aldrich, HT101128) or 4% Paraformaldehyde (Beyotime, p0099, Shanghai, China) for macroscopically metastatic nodule analysis.
item1768 The mice were divided into control group and ALKBH5-overexpressing group (9 mice per group). ALKBH5-overexpressing and control A549 cells (3 × 106 cells/mouse) in 200 uL PBS were intravenously (i.v.) injected into the lateral tail vein of mice. At every 5th day post-inoculation, TGF-Beta-1 (4 ug/kg body weight) was intraperitoneally (i.p.) injected to promote tumor cell metastasis. Eight weeks later, the mice were euthanized, and then their lungs and livers were taken out and fixed in Bouin's solution (Sigma Aldrich, HT101128) or 4% Paraformaldehyde (Beyotime, p0099, Shanghai, China) for macroscopically metastatic nodule analysis.
item1769 The mice were divided into control group and ALKBH5-overexpressing group (9 mice per group). ALKBH5-overexpressing and control A549 cells (3 × 106 cells/mouse) in 200 uL PBS were intravenously (i.v.) injected into the lateral tail vein of mice. At every 5th day post-inoculation, TGF-Beta-1 (4 ug/kg body weight) was intraperitoneally (i.p.) injected to promote tumor cell metastasis. Eight weeks later, the mice were euthanized, and then their lungs and livers were taken out and fixed in Bouin's solution (Sigma Aldrich, HT101128) or 4% Paraformaldehyde (Beyotime, p0099, Shanghai, China) for macroscopically metastatic nodule analysis.
item1770 Four-week-old male BALB/c nude mice were used for animal experiments. UM-UC3 cells (2×106 cells per mouse) stably overexpressing miR-5581-3p and NC were injected into the mice to establish the subcutaneous implantation model. Tumor size was measured by a caliper every week, and tumor volume was calculated by the formula: V = (width2×length×0.52). As for the tumor metastasis model, UM-UC3 cells (1×106 cells per mouse) were injected into each mouse via the tail vein. The subcutaneous implantation model used 8 nude mice, whereas the tumor metastasis model used 10 nude mice. Assessment of tumor size and observation of metastasis tumors were done via intraperitoneal inoculation with 15mg/mL, XenoLight D-luciferin Potassium Salt (100 uL; PerkinElmer) with the IVIS Spectrum animal imaging Platform (PerkinElmer) in every mouse. Eventually, mice were sacrificed for tumors and metastases.
item1771 Female C57BL/6 mice (6-8 weeks) were intraperitoneally injected with 2 ml 3% sterile sodium thioglycolate solution. After 3 days, the cells in the abdominal cavity were collected, centrifugated and maintained in DMEM with 10% (vol/vol) FBS.
item1772 Female C57BL/6 mice (6-8 weeks) were intraperitoneally injected with 2 ml 3% sterile sodium thioglycolate solution. After 3 days, the cells in the abdominal cavity were collected, centrifugated and maintained in DMEM with 10% (vol/vol) FBS.
item1773 .
item1774 .
item1775 A total of 5 × 106 stably transfected HGC-27 cells were subcutaneously injected into the right axillary fossa of nude mice. Tumor volume was measured every 3 days and calculated with the following formula: V = (L × W2)/2 cm2 (V, tumor volume; L, length; W, width). The mice were sacrificed at 3-4 weeks after injection, and the tumors were weighed. For the lung metastasis model, 5 × 106 stably transfected HGC-27 cells were injected into the tail veins of nude mice. Forty-five days later, the mice were sacrificed, and the lungs were dissected to examine the histopathological metastatic loci. The peritoneal dissemination ability of GC cells was evaluated via intraperitoneal injection. A total of 5 × 106 stably transfected HGC-27 cells in 500 uL of PBS were injected into the peritoneal cavity of BALB/c nude mice. Mice were carefully monitored until they were killed at 4 weeks, at which point peritoneal metastases were examined and recorded.
item1776 Control vector, TUSC7 knockout, FLI-06 treated H1975 cells (1*107) cells were suspended in 100 uL of serum-free DMEM medium (Hyclone, USA), mixed with matrix gel (Corning, USA), and then were injected subcutaneously. The changes in the tumor size were recorded every 3 or 5 days.
item1777 To study the effect of miR-30d on liver metastasis of PDACs, 5 × 106 cells in 50 uL of PBS were injected into the spleens of nude mice (6 mice per group). Anesthetized mice were injected intraperitoneally with D-luciferin (150 mg/kg) every other week and imaged using an IVIS 100 imaging system (Xenogen, CA, USA) 10?min after the injection. The mice were sacrificed and their liver metastases were checked by standard histological examination 8-9 weeks after injection.
item1778 .
item1779 .
item1780 HCC827 (3×106) cells suspended in 100 uL of PBS were injected into the left inguen of female Balb/c nude mice (body weight 18-20 g; age 6 weeks; Beijing Huafukang Bioscience Co., Inc.). When the tumor volumes reached 50-100 mm3 on the 10th posttransplantation day, the mice were randomized into four groups (10 mice per group) and were intragastrically administered vehicle (normal saline), crizotinib (25 mg/kg body weight), chidamide (5 mg/kg), or the combination of the two drugs daily for 21 days. The tumor volumes and body weights of the mice were measured every 3 days.
item1781 HCC827 (3×106) cells suspended in 100 uL of PBS were injected into the left inguen of female Balb/c nude mice (body weight 18-20 g; age 6 weeks; Beijing Huafukang Bioscience Co., Inc.). When the tumor volumes reached 50-100 mm3 on the 10th posttransplantation day, the mice were randomized into four groups (10 mice per group) and were intragastrically administered vehicle (normal saline), crizotinib (25 mg/kg body weight), chidamide (5 mg/kg), or the combination of the two drugs daily for 21 days. The tumor volumes and body weights of the mice were measured every 3 days.
item1782 .
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item1786 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1787 .
item1788 RIP1-Tag2 mice were purchased from NCI Mouse Repository (Bethesda, Rockville, MD, USA) and maintained in a C57BL/6N background.
item1789 .
item1790 .
item1791 OG2 (Oct4-GFP reporter) transgenic mice (CBA/CaJ × C57BL/6J) were original from Jackson Laboratory (Mouse strain datasheet: 004654).
item1792 OG2 (Oct4-GFP reporter) transgenic mice (CBA/CaJ × C57BL/6J) were original from Jackson Laboratory (Mouse strain datasheet: 004654).
item1793 .
item1794 Luciferase-labeled KYSE150 cells (5 × 106) were inoculated into the footpads of BALB/c nude mice (4-5 weeks old, 18-20 g) to establish the popliteal lymphatic metastasis model.
item1795 Luciferase-labeled KYSE150 cells (5 × 106) were inoculated into the footpads of BALB/c nude mice (4-5 weeks old, 18-20 g) to establish the popliteal lymphatic metastasis model.
item1796 .
item1797 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1798 .
item1799 .
item1800 .
item1801 .
item1802 For the subcutaneous implantation model, 1 × 106 cells were injected subcutaneously into the flank regions of female BALB/c nude mice (4-5 weeks).
item1803 IMQ-induced psoriatic model was constructed by applying 10 mg per ear 5% IMQ for 8 consecutive days, and 6 ug macrophage-specific control or hsa_circ_0004287 plasmid was topically applied every 2 days (5 mice per group per experiment).
item1804 IMQ-induced psoriatic model was constructed by applying 10 mg per ear 5% IMQ for 8 consecutive days, and 6 ug macrophage-specific control or hsa_circ_0004287 plasmid was topically applied every 2 days (5 mice per group per experiment).
item1805 .
item1806 Mice in the control group received an i.p. injections of 0.9% NaCl. Mice in the low, medium, and high Mn groups received i.p. injections of 12.5, 25, and 50 mg/kg MnCl2. The volume of administration was 5 mL/kg body weight. The injection was given daily for 2 weeks.
item1807 MKN74 cells (5×106/tumor) expressing shNC, shYTHDF1-1, or shYTHDF1-2 were suspended in ice-cold 100 ul PBS:Matrigel gel (1:1, v/v) (Corning, USA), and subcutaneously implanted into the right dorsal flank of 4-week-old NOD.
item1808 Luciferase-labeled rSKBR3 and MDA-MB-361 cells (1 × 107 cells) mixed with 1:1 Matrigel (Corning, 356237) were subcutaneously injected into the fat pads of mice. After a tumor was palpable, the mice were randomized into four groups (five mice per group), and they were treated with vehicle, trastuzumab (20 mg/kg, intraperitoneal administration), roblitinib (30 mg/kg, oral administration), or a combination of both drugs.
item1809 .
item1810 .
item1811 .
item1812 .
item1813 Nude mice were subcutaneously injected with 1 × 107 PADI2 depleted or IGF2BP1 depleted Ishikawa cells on the left flanks, and the corresponding control cells on the right flanks.
item1814 Four-week-old BALB/c female nude mice were purchased from Beijing HFK Bioscience Co.,LTD. The tumor masses were subcutaneously embedded in nude mice and the nude mice were randomly divided into two groups (n=5) when the tumor masses grew to 4 to 5mm in diameter. These two groups were control group and siALKBH5 group.
item1815 For the subcutaneous implantation model, 5 × 105 stable SLC7A11-knockdown HCCLM3 cells or SLC7A11-vector cells were injected subcutaneously into BALB/C nude mice.
item1816 .
item1817 .
item1818 .