Fig. 3.

m⁶A modification-mediated ferroptosis in HCC is autophagy-dependent. A Control siRNA and WTAP siRNA was transfected into Huh7 for12 h and then treated with erastin (10 μM) for 24 h. Total RNA was isolated for RNA-Seq. Clustering of Huh7 cells were demonstrated by microarray heat map. The significantly differentially expressed mRNAs were analysied by hierarchical cluster: white, no change; bright green, underexpression; bright orange, overexpression. B Enhanced Volcano was plotted by https://www.bioinformatics.com.cn, an online platform for data analysis and visualization. C Differentially expressed mRNAs were enriched by KEGG enrichment analysis in WTAP siRNA group (Erastin + Control siRNA, n = 3; Erastin + WTAP siRNA, n = 3). D The number of autophagosomes in Huh7 cells were observed by Transmission electron microscopic. Scale bars: 2.0 μm. E Huh7 cells transfected with WTAP siRNA and plasmid along with mRFP-EGFP-hLC3B-pKGX-Puro plasmid were treated with erastin (10 μM). Laser scanning confocal microscope was used to observe fluorescence intensity. Scale bars: 50 μm. F–H WTAP siRNA and plasmid were utilized to transfect Huh7 and HepG2 cells respectively followed by 10 μM erastin. Expression of autophagy-related genes was detected by Western blot. I Ferroptosis inducers (10 μM Erastin, 10 μM Sorafenib, and 2.5 μM RSL3) were applied to Huh7 for 24 h. MeRIP qPCR was used to detected the levels of m⁶A modification on autophagy-related genes. The mean ± SD was used as the value of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.