Fig. 6

FSH upregulates ALKBH5 mRNA expression via CREB signaling. HUVECs were treated with varying concentrations of FSH for different time intervals, and ALKBH5 mRNA levels were assessed by qRT-PCR (A, B). HUVECs were treated with PI3K inhibitor wortmannin (WM, 1 μM), MEK inhibitor PD98059 (PD, 20 μM), PKA inhibitor H89 (10 μM), or CREB inhibitor 666–15 (10 μM), followed by treatment with FSH (100 mIU/mL) for 24 h. Protein expression was analyzed by WB (C). HUVECs were treated with different concentrations of FSH (10–100 mIU/mL) for varying durations (12–72 h), and the protein expression of total CREB and phosphorylation of CREB at Ser129/133 was detected by WB (D, E). HUVECs transfected with CREB siRNA were treated with FSH (100 mIU/mL) for 48–72 h, and protein expression was analyzed by WB (F, G). A schematic overview depicts the predicted p-CREB S129/133 binding sites within the ALKBH5 promoter region, with vertical bars indicating binding sites (H). HUVECs were treated with or without FSH (100 mIU/mL) for 24 h, and ChIP-qPCR confirmed the binding of p-CREB to the ALKBH5 promoter (I). A schematic diagram illustrates deletion plasmids containing different ALKBH5 promoter fragments upstream of the firefly luciferase reporter gene in the pGL4.10 vector (left). Following transfection of deletion plasmids and CREB overexpression (CREB O/E) or negative control plasmid (NC) into 293 T cells for 24 h, luciferase activity was measured (J, **p < 0.01 versus NC + WT, #p < 0.05, ##p < 0.01 versus CREB O/E + WT). Statistical analysis was conducted using one-way ANOVA with Dunnett’s multiple comparisons test (A, B) and Sidak’s multiple comparisons test (J). Data were analyzed by two-way ANOVA with Sidak’s multiple comparisons test (I). n ≥ 3, *p < 0.05, **p < 0.01