Fig. 3
FTO modifies the m6A level of FAP in an m6A-YTHDF2-dependent manner. a FAP was identified as a downstream target of FTO-mediated m6A modification by the human m6A epitranscriptomic microarray analysis. Venn diagram illustrating the overlap of significantly differentially expressed genes. b Western blot assay of FTO and FAP protein expression levels in 20 paired NSCLC tissues and the paired para-cancerous tissues. c Correlation analysis of the protein expression levels of FTO and FAP in NSCLC tumor samples. d The relative mRNA expression levels of FAP in FTO-knockdown or FTO-overexpressing NSCLC cell lines. e The relative protein expression levels of FAP in FTO-knockdown or FTO-overexpressing NSCLC cell lines. f Wild-type or m6A consensus sequence mutant FAP was fused to a firefly luciferase reporter. Mutation of the m6A consensus sequences was generated by replacing adenosine (A) with cytosine (C). g-h Relative luciferase activity of the wild-type and mutant FAP reporter vectors in FTO-overexpressing NSCLC cells. i RIP assay using an anti-FTO antibody or IgG to detect the binding to FAP in H1299 cells. IgG was used as the negative control. j-l MeRIP qRT‒PCR assay, using an anti-m6A antibody or IgG to detect the binding to FAP in H1299 cells. IgG was used as the negative control. m The mRNA expression levels of FAP were analyzed by qRT‒PCR in H1299 cells transfected with the indicated constructs. n RIP assay, using an anti-YTHDF2 antibody or IgG to detect the binding to FAP in H1299 cells. IgG was used as the negative control. o The relative mRNA expression ratio of FAP was analyzed by qRT‒PCR in actinomycin D-treated H1299 cells at various time points (2, 4, 6, and 8 h). p The relative mRNA expression levels of FAP in H1299 cells transfected with the indicated constructs. Data information: Data are shown as the mean ± SDs. In all relevant panels, *P < 0.05; **P < 0.01; ***P < 0.001