Fig. 3. m⁶A regulates the mRNA stability and translation of PDK4 in cancer cells.

a Cells were transfected with pGL3-Basic-PDK4-luc reporter and pRL-TK plasmid for 24 h. Results were presented as the ratios between the activity of the reporter plasmid and pRL-TK. b The relative levels of nuclear versus cytoplasmic PDK4 mRNA in wild-type and Mettl3^Mut/- cells, or sh-control and sh-Mettl3 Huh7 cells. c After treatment with Act-D to inhibit transcription, the precursor mRNA levels of PDK4 were checked in wild type and Mettl3^Mut/- cells. d After treatment with Act-D for the indicated times, the mature mRNA levels of PDK4 were checked in wild type and Mettl3^Mut/- cells. e HeLa cells were pre-transfected with vector control or Mettl3 construct for 24 h and then further treated with CHX (10 μg/ml) or MG-132 (5 μM) for 6 h, the expression of PDK4 was detected by western blot analysis (left) and quantitatively analyzed (right). f Cells were treated with 10 μg/ml CHX for the indicated time periods, the expression of PDK4 was detected by western blot analysis (left) and quantitatively analyzed (right). g Wild-type or Mettl3^Mut/- HeLa cells were transfected with pmirGLO-PDK4 reporter for 24 h. The translation outcome was determined as a relative signal of F-luc divided by R-luc, the mRNA abundance was determined by qRT-PCR of F-luc and R-luc, and the translation efficiency of PDK4 is defined as the quotient of reporter protein production (F-luc/R-luc) divided by mRNA abundance²⁸. h qRT-PCR checked the mRNA levels of PDK4 in non-ribosome portion (<40S), 40S, 60S, 80S, and polysome fractions in wide type and 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) and (c) (p < 0.0001 for all comparisons), and two-way ANOVA for (b) (p > 0.05), and by one-way ANOVA for (e) (p = 0.005) and (h) (p < 0.0001 for all comparisons).