Journal: PLoS ONE

Article Title: Tocilizumab does not block interleukin-6 (IL-6) signaling in murine cells

doi: 10.1371/journal.pone.0232612

Figure Lengend Snippet: (A) Human U-937 cells were pretreated with tocilizumab as indicated for 30 min and then stimulated with 10 ng/ml hIL-6 for 15 min. Phosphorylation of STAT3 was determined by western blot. Quantification of three independent experiments and one representative western blot are shown. (B-D) The experiments were performed as described under (A) but with murine RAW264.7 cells and mIL-6 (B), with human HepG2 cells and hIL-6 (C) and with murine AML-12 cells and mIL-6 (D). Statistical significance was analyzed with one-way ANOVA followed by Dunnett's multiple comparisons test (**: p<0.01; ***: p < 0.001; ns: not significant).

Article Snippet: The human monocyte cell line U-937 (CRL-1593.2) and the murine macrophage cell line RAW264.7 (SC-6003) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human hepatocyte cell line HepG2 (ACC 180) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Phospho-proteomics, Western Blot

Journal: British Journal of Pharmacology

Article Title: Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

doi: 10.1111/bph.13016

Figure Lengend Snippet: Effects of tamoxifen, raloxifene, toremifene and lovastatin on DiI-LDL uptake by MOLT-4 cells, lymphocytes and HepG2 cells. (A) MOLT-4 cells were treated with DiI-LDL and vehicle, 5 μM tamoxifen (TAM), raloxifene (RAL) or toremifene (TOR), 1 μM lovastatin (LOV) or the combinations of these drugs for 24 h as indicated. Data (mean ± SEM) correspond to five independent experiments and are expressed as % of the corresponding condition without any drug added. (B) Lymphocytes from 10 male donors were treated as indicated for MOLT-4 cells. Data (mean ± SEM) are expressed as % of the condition without any drug added of the same cell preparation. (C) HepG2 cells were treated as indicated for MOLT-4 cells and lymphocytes. Data (mean ± SEM) correspond to five independent experiments and are expressed as % of the corresponding condition without any drug added. Statistical analyses were performed by two-way RM anova and post hoc by the Student–Newman–Keuls test. ●●P < 0.01, ●●●P < 0.001 between presence and absence of LOV; *P < 0.05, ***P < 0.001 versus control; ##P < 0.01, ###P < 0.001 versus RAL; §§P < 0.01 versus TOR. For simplicity, the statistical differences between the conditions within the presence of LOV were omitted.

Article Snippet: HepG2 human hepatocytes (ATCC CRL-11997) and MOLT-4 human lymphoblastoid cells (ATCC CLR 1582) were obtained from the American Type Culture Collection.

Techniques: Control

Journal: Nutrition & Metabolism

Article Title: METTL3 exacerbates insulin resistance in hepatocytes by regulating m 6 A modification of cytochrome P450 2B6

doi: 10.1186/s12986-023-00762-z

Figure Lengend Snippet: High global m 6 A methylation change in NAFLD models. A comparison of the mice body size, the mice liver size ( A ) and the mice weight ( B ) between the control group (blue) and the HFD group (red). The mice in HFD group (n = 16) were feeding with high fat diet for 16 weeks, while the mice in the control group were feeding with normal chow diet (n = 15). HE staining ( C ) (scale bars, 20 μm) of livers between the HFD and control group. The IPITT ( D ) and the IPGTT (E) tests in the HFD and control mice, the concentration of the injected insulin and glucose is 0.65 U/kg and 2.0 g/kg, respectively. LC–MS analysis ( F ) of m 6 A level in HFD mice liver and steatosis HepG2 cells (induced by 1 mmol/L FFA). Dot blot analysis ( G ) in the mice liver and in the steatosis HepG2 cells incubated with m 6 A antibodies. HE staining (scale bars, 20 μm) and the dot blot analysis (H) in the liver samples from NAFLD patients (n = 3) or CD patients (n = 3)

Article Snippet: The human hepatic cell line HepG2 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan; China).

Techniques: Methylation, Comparison, Control, Staining, Concentration Assay, Injection, Liquid Chromatography with Mass Spectroscopy, Dot Blot, Incubation

Journal: Nutrition & Metabolism

Article Title: METTL3 exacerbates insulin resistance in hepatocytes by regulating m 6 A modification of cytochrome P450 2B6

doi: 10.1186/s12986-023-00762-z

Figure Lengend Snippet: METTL3 increased in hepatic IR and interference of METTL3 attenuated IR induced by high fat. PT-qPCR analysis of all writers (Alkb5 and Fto) and erasers (Mettl14, Mettl3, Virilizer, Wtap) mRNA expression in livers from HFD mice (red) and control mice(black) ( A ). Representative western blot analysis of all writers and erasers expression in HepG2 cells between FFA group and Control group ( A ). Representative western blot analysis and quantification of Mettl3 expression in HepG2 cells between FFA group and NC group ( C ). Glucose consumption ( D ) and glucose uptake ( E ) analysis in HepG2 cells, it was divided into six groups, disposed with or without insulin; with or without FFA; with or without Mettl3 SiRNA. Representative western blot analysis and quantification of Glut2 expression in HepG2 cells disposed with insulin (10 −7 mmol/L insulin for 4 h) between METTL3 SiRNA group and Control group ( F )

Article Snippet: The human hepatic cell line HepG2 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan; China).

Techniques: Expressing, Control, Western Blot

Journal: Nutrition & Metabolism

Article Title: METTL3 exacerbates insulin resistance in hepatocytes by regulating m 6 A modification of cytochrome P450 2B6

doi: 10.1186/s12986-023-00762-z

Figure Lengend Snippet: Elevated CYP2B6 m6A modification and upregulation of CYP2B6 expression were identified in NAFLD model. A mouse m 6 A epitranscriptomic microarray test of the NAFLD mice liver between HFD and control group, of all the mRNA detected, 108 hypermethylated and 212 hypomethylated genes were changed significantly in HFD-fed group ( A ). The GO and pathway analysis of the methylated mRNA showed metabolic process were enriched ( B ) and in Type II diabetes mellitus, insulin signaling pathway and adipocytokine signaling pathway ( C ). Between all the methylated mRNA, CYP450 family genes were plotted in the heatmap, the Merip-qPCR analysis showed Cyp2b10 (ENSMUST00000005477) m 6 A level was elevated ( D ) in the HFD group. IHC staining and quantitative analysis of Cyp2b10 in mice liver from HFD group and control group. Scale bars, 20 μm ( E ). A RT-qPCR analysis of Cyp2b6 between HFD group and control group and a western blot analysis of CYP2B6 (Homologous to mouse Cyp2b10 in HepG2. A western blot analysis of CYP2B6 in patients’ liver between the NAFLD group (n = 4) and the CD group (n = 4)

Article Snippet: The human hepatic cell line HepG2 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan; China).

Techniques: Modification, Expressing, Microarray, Control, Methylation, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Nutrition & Metabolism

Article Title: METTL3 exacerbates insulin resistance in hepatocytes by regulating m 6 A modification of cytochrome P450 2B6

doi: 10.1186/s12986-023-00762-z

Figure Lengend Snippet: METTL3 upregulated CYP2B6 m 6 A modification and positively regulate CYP2B6 expression. Merip-qPCR analysis of the m 6 A level of CYP2B6 between the METTL3 down-expression group (infected with METTL3 SiRNA) and the METTL3 over-expression group (transfected with LV5 lentivirus) ( A ). RIP assay was performed using anti-METTL3 antibodies ( B ). RT-qPCR analysis and western blot analysis of CYP2B6 expression in HepG2 between the group of METTL3 down-expression (METTL3 SiRNA) and the group of METTL3 over-expression (METTL3 LV5 lentivirus) ( C ). Representative western blot analysis and quantification of CYP2B6 expression in HepG2 from FFA + METTL3 −/− group, NC + METTL3 −/− group and FFA group ( D ). Dot blot test in mice liver from HFD + STM2457 group, HFD group, HFD + 0.9% NaCL group and HFD + DMSO group ( E ). Both IPITT and IPGTT results showed the increased insulin sensitivity in STM2457 group ( F / G ). Representative western blot analysis and quantification of CYP2B6 expression in HepG2 from HFD + STM2457 group and control group ( H )

Article Snippet: The human hepatic cell line HepG2 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan; China).

Techniques: Modification, Expressing, Infection, Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Dot Blot, Control

Journal: Nutrition & Metabolism

Article Title: METTL3 exacerbates insulin resistance in hepatocytes by regulating m 6 A modification of cytochrome P450 2B6

doi: 10.1186/s12986-023-00762-z

Figure Lengend Snippet: Overexpression of CYP2B6 antagonize METTL3-mediated hepatic insulin resistance by regulating pIRS/IRS expression. Representative western blot analysis and quantification of IRS and pIRS expression in HepG2 from FFA group, METTL3 −/− group and FFA + METTL3 −/− group ( A ). Representative western blot analysis and quantification of IRS and pIRS expression in HepG2 from FFA group, CYP2B6 +/+ group and FFA + CYP2B6 +/+ group ( B ). Representative western blot analysis and quantification of IRS and pIRS expression in mice liver from HFD + 0.9% NaCL group, HFD + DMSO group and HFD + STM2457 group ( C ). A schematic model related with the present study ( D )

Article Snippet: The human hepatic cell line HepG2 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan; China).

Techniques: Over Expression, Expressing, Western Blot