Figure 1.

METTL3 expression was elevated and correlated with high m⁶A levels in the M phase. (A) Venn diagram illustrated the upregulated and downregulated genes differentially expressed in the M phase compared to other cell cycle phases (G1 phase, S phase and G2 phase). (B) The protein levels of METTL3, METTL14, FTO, ALKBH5 and WTAP were analyzed by Western blotting. ACTIN served as a loading control. (C) The relative mRNA levels of METTL3, METTL14, FTO and ALKBH5 were analyzed by qPCR. **** P < 0.0001 (one-way ANOVA analysis) for the indicated comparisons. Relative mRNA expression levels were normalized by 18S rRNA. Data are presented as the mean ± SD for n = 3 replicate cultures. (D) Global m⁶A mRNA methylation levels in various HeLa cell cycle phases were determined by m⁶A enzyme-linked immunosorbent assays. m⁶A RNA methylation quantification was performed based on the standard curve. * P < 0.05, ** P < 0.01 (one-way ANOVA analysis) for the indicated comparisons. (E) Quantification of m⁶A enrichment in mRNA from control and siMETTL3 cells in the M phase. The knockdown efficiency of METTL3 in HeLa cells was confirmed by Western blotting indicated in the upper panel. Quantification of m⁶A was detected by the m⁶A RNA methylation quantification kit. Data shown are mean ± SD for n = 3 replicate cultures, * P < 0.05 compared to negative control. (The western blotting data are representative of n=3 independent experiments).