Journal: Advanced Science

Article Title: IL6 Derived from Macrophages under Intermittent Hypoxia Exacerbates NAFLD by Promoting Ferroptosis via MARCH3‐Led Ubiquitylation of GPX4

doi: 10.1002/advs.202402241

Figure Lengend Snippet: IL‐6 derived from macrophages under IH induced ferroptosis and lipid accumulation in hepatocytes. A–D) RNA‐seq data of HepG2 cells treated with CM‐control or CM‐IH (24 h). A) Heatmap, B) volcano map, C) PPI analysis, and D) enrichment analysis of DEGs. E) IL6 mRNA level in macrophages evaluated by q‐PCR. F) IL6 concentration in the macrophage supernatant determined by ELISA. G) Level of 4‐HNE determined using IF (scale bar, 100 µm). H) Level of lipid ROS determined using flow cytometry. I) Mitochondrial membrane potential was assessed using JC‐1 staining, which revealed mitochondria with high (increased PE) or low (increased FITC) membrane potential. J) Mitochondrial structure in HepG2 and LO2 cells under an electron microscope (scale bar, 500 nm). Levels of K) MDA and L) GSH in hepatocytes. M) Oil Red O staining of HepG2 and LO2 cells from the DMSO and IL6 NAbs groups (200 ng mL −1 , 24 h). N) TG level in HepG2 or LO2 cells treated with DMSO or IL6 NAbs. All bar charts shown represent the mean ± SEM, * p < 0.05, ** p < 0.01, by t ‐test.

Article Snippet: IL6 neutralizing antibodies (IL6 NAbs, 200 ng mL −1 ) (SinoBiological, China) were added to CM‐IH to inhibit IL6 in the CM‐IH+IL6 NAbs group.

Techniques: Derivative Assay, RNA Sequencing Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Membrane, Staining, Microscopy

Journal: Advanced Science

Article Title: IL6 Derived from Macrophages under Intermittent Hypoxia Exacerbates NAFLD by Promoting Ferroptosis via MARCH3‐Led Ubiquitylation of GPX4

doi: 10.1002/advs.202402241

Figure Lengend Snippet: IL6‐induced degradation of GPX4 protein in hepatocytes was dependent on MARCH3. A) GPX4 mRNA levels in HepG2 cells evaluated by q‐PCR. B) GPX4 protein levels in HepG2 cells determined by western blot analysis (20 × 10 −6 m MG132, 100 µg mL −1 CHX). C) Potential E3 ligases of GPX4 predicted by UbiBrowser. D) Transcription levels of MARCH1, MARCH3, MARCH8, and MARCH11 determined via RNA‐seq in HepG2 cells. E,F) Levels of GPX4 and MARCH3 in HepG2 cells treated with different interventions were evaluated by western blot analysis. G) Interaction between MARCH3 and GPX4 was confirmed by co‐IP. GPX4 ubiquitination level in cells with H) exogenous overexpression or I) endogenous knockdown of MARCH3. J) Levels of nGPX4(nucleus GPX4), cGPX4(cytoplasm GPX4) and mGPX4(mitochondria GPX4) in HepG2 cells treated with different interventions were evaluated by western blot analysis. K) Intersection of DEGs and predicted transcription factors targeting MARCH3 genes was shown using a Venn diagram. L) Levels of RUNX1, MARCH3, and GPX4 in HepG2 cells treated with CM‐IH and siRUNX1. All bar charts shown represent the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant, A by t ‐test, B‐K by ANOVA.

Article Snippet: IL6 neutralizing antibodies (IL6 NAbs, 200 ng mL −1 ) (SinoBiological, China) were added to CM‐IH to inhibit IL6 in the CM‐IH+IL6 NAbs group.

Techniques: Western Blot, RNA Sequencing Assay, Co-Immunoprecipitation Assay, Over Expression, Knockdown