Figure 4A Gene Set Enrichment Analysis (GSEA) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3-knockdown HepG2, SMMC-7721 and Bel-7402 cells with the C5. biological process symbol set. (p < 0.001). The KEGG analysis shows that FOXO3 involved in hypoxia and autophagy. C Protein level of FOXO3 in METTL3-knockdown HCCs under hypoxia for 48h. D Half-life of FOXO3 mRNA in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with actinomycin D. E Half-life of FOXO3 protein in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with cycloheximide. Quantification of the protein optical density by Image J in E-1. F Analysis of FOXO3 mRNA in non-ribosome portion (<40S), 40S, 60S, 80S and polysome for the METTL3-knockdown Bel-7402 cells compare to control cells under hypoxia for 48h. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. G Knockdown of YTHDF1 and YTHDF2 in Bel-7402 cells by lentiviral shRNAs. H Knockdown of YTHDF1 but not YTHDF2 abolished the increase of FOXO3 protein level in METTL3-overexpressing Bel-7402 cells. Quantification of the protein optical density by Image J in H-1 and H-2. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. I RNA level of FOXO3 in YTHDF1-knockdown Bel-7402 cells by RT-PCR. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. J The m6A-IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3. The YTHDF1 binding site locates at the 3'UTR of FOXO3. K 800 bp of FOXO3 3'UTR containing the m6A modification site was cloned into luciferase reporter vector. Mutation of m6A consensus sequence was generated by replacing adenosine with thymine. L Relative luciferase activity of the wild-type and mutant form of FOXO3 3'UTR reporter vectors in Bel-7402 cells with various levels of METTL3 and YTHDF1. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1-RIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. O Correlation analysis of the RNA expression levels of METTL3 and FOXO3 in liver tumor cohort (Kim-90) by Pearson. P Correlation analysis of the RNA expression levels of YTHDF1 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.