Fig. 2
FTO promotes expression of AML1-ETO as an m6A demethylase by repressing YTHDF2-mediated AML1-ETO mRNA decay. A, B The m6A abundance in mRNA detected by m6A dot blotting (A) and the expression levels of FTO and AML1-ETO by western blotting (B) in SKNO-1 or Kasumi-1 AML cells with forced expression of wild-type FTO (wt-FTO), mutant FTO (mut-FTO), or mock vectors; Methylene blue (MB) represents the loading control of RNA samples. C, D The m6A dot blotting (C) and western blotting (D) in SKNO-1 or Kasumi-1 AML cells transfected with FTO shRNA or scramble shRNA (shNS) vectors. E, F Effects of FB23-2 treatment for 72 h on the m6A abundance in mRNA by m6A dot blotting (E) and expression levels of AML1-ETO detected by western blotting (F) in SKNO-1 and Kasumi-1 cells. G RIP-qPCR showing the interaction between AML1-ETO mRNA transcripts and FTO protein in SKNO-1 and Kasumi-1 cells. H, I The levels of m6A in AML1-ETO transcripts assessed by gene-specific m6A qPCR assay in SKNO-1 and Kasumi-1 cells transduced with wt-FTO, mut-FTO, or mock vector (H); or with FTO shRNA or shNS vectors (I). J–L The mRNA half-life (t1/2) of AML1-ETO transcripts in SKNO-1 and Kasumi-1cells transduced with wt-FTO, mut-FTO, or mock vector (J); or with FTO shRNA or shNS vectors (K) or treated with DMSO or FB23-2 for 72 h (L). M RIP-qPCR showing the interaction between AML1-ETO mRNA transcripts and YTHDF2 protein in SKNO-1 and Kasumi-1 cells. N, O Increased expression of AML1-ETO after YTHDF2 knockdown assessed by qPCR (N) and western blotting (O) in SKNO-1 and Kasumi-1 cells. P Increased mRNA half-life (t1/2) of AML1-ETO transcripts in SKNO-1 and Kasumi-1 cells after YTHDF2 knockdown