Fig. 3.

Reduced ferroptosis by m⁶A modification inhibition is associated with autophagy inactivation. (A) FTO plasmid was transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h. Total RNA was isolated for RNA-Seq. Clustering of HSC-LX2 cells were demonstrated by microarray heat map. The significantly differentially expressed mRNAs were analysied by hierarchical cluster: gray, no change; bright blue, underexpression; bright red, overexpression (FTO plasmid, n = 3; Control vector, n = 3). (B) Differentially expressed mRNAs were enriched by KEGG enrichment analysis in FTO plasmid group (Control vector, n = 3; FTO plasmid, n = 3). (C) The levels of m⁶A modification in autophagy-related gene were determined by MeRIP qPCR (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 3 in every group). (D) METTL4 shRNA or FTO plasmid transfected into HSC-T6 and HSC-LX2 cells were treated with erastin (10 μM) for 24 h. Western blot showed the protein expression of BECN1, ATG3, ATG4A, ATG7, ATG9A, ATG5-ATG12 and ATG16L1 (n = 3 in every group). (E) Western blot was used to determine the expression of LC3-I/II and p62 (n = 3 in every group). (F) METTL4 shRNA or FTO plasmid with CMV-TurboRFP- EGFP-LC3-PGK-Puro plasmid were transferred into HSC-LX2 cells by erastin (10 μM) treatment for 24 h. The fluorescence spots were detected. Representative photographs were showed. Scale bars: 50 μm. (G) HSC-LX2 cells transfected with FTO plasmid or control vector by erastin (10 μM) treatment for 24 h. Transmission electron microscopy was used to examine the autolysosomes or autophagosomes. Representative photographs were showed. Scale bars: 0.2 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)