Fig. 4

FSH increases FOXM1 expression through ALKBH5-mediated m6A demethylation. HUVECs were treated with FSH (100 mIU/mL) for 24 h, and the relative activity of the FOXM1 promoter firefly luciferase reporter (1 μg) was measured (A). The proportion of m6A in total RNA and total mRNA was assessed by colorimetry (B), and FOXM1 mRNA m6A levels were analyzed by MeRIP (C). HUVECs were treated with varying concentrations of FSH (10–100 mIU/mL) for different durations (12–72 h), and protein expression levels of ALKBH5, FTO, METTL3, and METTL4 were detected by WB (D, E). HUVECs were transfected with ALKBH5 shRNA or ALKBH5 overexpression (O/E) constructs and treated with FSH (100 mIU/mL) for 24 h, after which FOXM1 mRNA m6A levels were detected by MeRIP (F). Following transfection with ALKBH5 shRNA, ALKBH5 O/E, or the ALKBH5 H204A mutant, HUVECs were treated with FSH (100 mIU/mL) for 24 h, and the indicated protein expressions were measured by WB (G), with FOXM1 mRNA levels determined by qRT-PCR (H–J). HUVECs transfected with ALKBH5 shRNA or ALKBH5 O/E were treated with FSH (100 mIU/mL) for 72 h, and EndMT-related protein expression was analyzed by WB (K, L). M Following ALKBH5 shRNA transfection, HUVECs were treated with FSH (100 mIU/mL) for 72 h, and cell migration was assessed using a wound-healing assay over 24 h. N Quantification of the wound-healing assay. Data were analyzed using an unpaired t-test (A, C) and one-way ANOVA with Sidak’s multiple comparisons test (F, H, I, J, N). Statistical analysis was conducted using two-way ANOVA with Sidak’s multiple comparisons test (B). n ≥ 3, *p < 0.05, **p < 0.01. Scale bars: 250 μm (M)