Figure 4. Overexpression of LNK impairs insulin signaling and induces apoptosis of granulosa cells. (A) Representative western blots of phosphorylated or total FOXO3 and AKT in LNK overexpression and silence granulosa cells. Ovarian granulosa cells were transfected for 48 hours and then starved for 12 hours with 1% FBS culture medium. After that, 100 nM insulin was added to the culture medium for 30 minutes, then cells were harvested for subsequent experiments. (B, C) Densitometry analysis of phosphorylation of AKT and FOXO3 in granulosa cells. The levels of phosphorylated AKT/AKT and phosphorylated FOXO3/FOXO3 were inhibited by overexpression of LNK in KGN cells. Results are mean±SD. *p < 0.05 vs. data of control group. (D) Immunofluorescence: pc LNK: FOXO3 dephosphorylated and its expression was increased in the nucleus. siLNK: FOXO3 was mainly expressed in the cytoplasm. Scale bar, 20μm. (E) Apoptosis detection of KGN was analyzed by TUNEL staining. Scale bar, 200μm. (F) Comparison of early and late apoptosis rate of KGN cells by Annexin V FITC-A flow cytometry analysis. Values are shown as means±SD, n=3, *p<0.05 vs. Con. (G) Comparison of FSHR. The KGN cells were transfected for 48 hours, then cells were harvested for RNA extraction. Values are shown as means ± SD, n=3, *p<0.05 vs. Con.