Fig. 5. Factors involved in m⁶A regulated expression of PDK4.

a RIP-qPCR analysis of PDK4 mRNA in wild type HeLa cells by use of antibody of YTHDF2, YTHDF3, and IGF2BP1~3. b IGF2BP3 RIP-qPCR analysis of PDK4 mRNA in wild type or Mettl3^Mut/- HeLa cells. c Wild type or Mettl3^Mut/- HeLa cells were transfected with si-NC or si-IGF2BP3 for 24 h, the expression of PDK4 was checked by western blot analysis (left) and quantitatively analyzed (right). d, e HeLa cells were transfected with si-NC, si-IGF2BP3, pcDNA-PDK4-5′UTR-WT (d) or pcDNA-PDK4-5′UTR-Mut1 (e) for 24 h and then further treated with Act-D for the indicated times. The mRNA of PDK4 was checked by qRT-PCR. f YTHDF1 RIP-qPCR analysis of PDK4 mRNA in wild type or Mettl3^Mut/- HeLa cells. g Binding of YTHDF1 with the 5′UTR or 3′UTR in wild type or Mettl3^Mut/- HeLa cells were analyzed by YTHDF1 RIP-qPCR using fragmented RNA. h Wild type or Mettl3^Mut/- HeLa cells were transfected with vector or YTHDF1 construct for 24 h, the expression of PDK4 was checked by western blot analysis (left) and quantitatively analyzed (right). i HeLa cells were transfected with vector, YTHDF1 construct, pcDNA-PDK4-5′UTR-WT, and pcDNA-PDK4-5′UTR-Mut1 for 24 h, the expression of PDK4 was checked by western blot analysis (left) and quantitatively analyzed (right). j eEF-1 and eEF-2 RIP-qPCR analysis of PDK4 mRNA in wild type or Mettl3^Mut/- HeLa cells. Data are presented as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, NS, no significant, by two-tailed unpaired Student’s t test for (a) (p < 0.0001) and (g) (p < 0.0001 and p = 0.11, respectively), and two-way ANOVA for (b) (p < 0.0001 and p = 0.004, respectively), (c, d) (p < 0.0001 for all), (f) (p = 0.0003 and p = 0.001, respectively), (h–j).