Fig. 4

YTHDF2 mediated the DDIT4 mRNA degradation in an m6A-dependent manner. A IGV plots showed the m6A and YTHDF2 binding site and the change of RNA-seq peaks at DDIT4 mRNAs in CAL-62. B The sequence represents the fragments captured in MeRIP-seq, which was co-localized with predicted sites. C The m6A peaks motifs are RRACH (R = G/A, H = A/C/U). D and E Relative luciferase activity of DDIT4 5’UTR with wild-type or mutated m6A sites after YTHDF2 knockdown and overexpression in CAL-62 and BHT101 cells. Renilla luciferase activity was measured and normalized to firefly luciferase activity. F The total RNA m6A level after DAA treatment was detected by m6A RNA dot blot assay and compared with DMSO treatment, with methylene blue staining as a loading control. G qPCR was used for the detection of DDIT4 mRNA levels after DAA treatment. H MeRIP-RT-qPCR detection of m6A level alterations of DDIT4 after METTL3 knockdown in CAL-62 cells. I RT-qPCR analysis of the decay rate of DDIT4 mRNA after actinomycin D (5 µg/mL) treatment in CAL-62 and BHT101 cells with YTHDF2knockdown or overexpression. Error bars represent the SD from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001