Figure 4.

Differentially methylated FOXO3 transcript was regulated via the YTHDF2‐mediated decay pathway in the luteinized GCs of the controls. A, m⁶A peaks distribution across 3’‐UTR of the FOXO3 transcript. The Y‐axis shows read number. Blue boxes indicate exons, and blue line indicates introns. B, qRT‐PCR analysis of m⁶A peak region and non‐peak region in the 3’‐UTR of the FOXO3 transcript. Bars represent means ± SD, n = 5. *P < 0.05 for the differences between the indicated groups. C, Schematic representation of the position of m⁶A motif in the 3’‐UTR of the FOXO3 transcript. D, The mRNA level of FOXO3 in the YTHDF2‐knockdown cells. Bars represent means ± SD, n = 5. *P < 0.05 for the differences between the indicated groups. E, Western blot analysis of FOXO3 total protein level in the YTHDF2‐knockdown cells. F, Wild‐type or m⁶A motif mutant (A‐to‐T mutation) FOXO3‐3’‐UTR fused with Renilla luciferase reporter. G, Relative luciferase activity of the FOXO3‐3’‐UTR with wild‐type or mutant m⁶A motif after cotransfection with negative control siRNA or FTO‐siRNA Renilla luciferase activity was normalized to firefly luciferase activity. Bars represent means ± SD, n = 3. *P < 0.05 vs the control siRNA group