Fig. 3. FTO downregulation increases m⁶A of MYC mRNA, thereby enhancing c-Myc expression.

A m⁶A RNA sequencing (m⁶A-seq) of H322 cells with or without FTO depletion was conducted. The proportions of m⁶A peak distribution in the 5′ untranslated region (5′UTR), start codon, coding region (CDS), stop codon, and the 3′ untranslated region (3′UTR) across the entire set of mRNA transcripts were calculated. B m⁶A RNA sequencing (m⁶A-seq) of H322 cells with or without FTO depletion was conducted. Reactome pathway analysis of genes with increased m⁶A abundance (fold-change>4) in H322 cells with FTO depletion was performed. C m⁶A RNA sequencing (m⁶A-seq) of H322 cells with or without FTO depletion was conducted. The m⁶A enrichment on MYC mRNA is shown. A solid triangle indicates the region for qPCR analyses. D Methylated RNA in H322 (left) and H358 (right) cells with or without FTO depletion was immunoprecipitated with an m⁶A antibody followed by qPCR analyses with primers against MYC mRNA. Data represent the means ± SD of triplicate samples. **P < 0.01, ***P < 0.001, and ****P < 0.0001. E RIP analyses of H322 and H358 cells were performed with an anti-FTO antibody followed by qPCR analyses with primers against MYC mRNA. Data represent the means ± SD of triplicate samples. ****P < 0.0001. F Luciferase vectors with WT or mutated m⁶A nucleotides in the MYC gene were transfected into 293T cells with or without FTO depletion. Luciferase activity was measured. Data represent the means ± SD of triplicate samples. ****P < 0.0001. G H322 and H358 cells with or without FTO depletion were analyzed by immunoblotting analyses with the indicated antibodies.