Fig. 6
FTO promotes the expression of IGFBP2 in an m6A-dependent manner. A, B Expression of IGFBP2 assessed by qPCR (A) and western blotting (B) in SKNO-1 or Kasumi-1 cells transduced lentivirally with wild-type FTO (wt-FTO), mutant FTO (mut-FTO), or mock vectors. C, D Decreased expression of IGFBP2 detected by qPCR (C) and western blotting (D) in SKNO-1 and Kasumi-1 cells after knockdown of FTO by shRNAs. E, F Expression of IGFBP2 analyzed by qPCR (E) and western blotting (F) in SKNO-1 or Kasumi-1 cells after FB23-2 treatment for 72 h. G RIP-qPCR showing the interaction between IGFBP2 mRNA transcripts and FTO protein in SKNO-1 and Kasumi-1 cells. H, I The levels of m6A in IGFBP2 transcripts assessed by gene-specific m6A qPCR assay in SKNO-1 and Kasumi-1 cells transduced with wt-FTO, mut-FTO, or control vector (H); or with FTO shRNA or shNS vectors (I). J Dual luciferase reporter assays for the effect of wild-type or mutant FTO on the relative luciferase activity of psiCHECK2-IGFBP2-3′-UTR with either wild-type or mutant m6A sites. K, L The mRNA half-life (t1/2) of IGFBP2 transcripts in SKNO-1 and Kasumi-1 cells transduced with FTO shRNA or shNS vectors (K) or treated with DMSO or FB23-2 for 72 h (L). M–O RIP-qPCR showing the interaction between IGFBP2 mRNA transcripts and YTHDF1 (M), YTHDF2 (N) and YTHDF3 (O) protein in SKNO-1 and Kasumi-1 cells. P, Q Increased expression of IGFBP2 after YTHDF1, YTHDF2 and YTHDF3 knockdown analyzed by qPCR (P) and western blotting (Q) in SKNO-1 and Kasumi-1 cells