Fig 1: Effects of Klf4 overexpression on cell viability and proliferation of preovulatory GCs. Cell viability was determined by (A) CCK-8 cell counting assay (ALX-850-039-0100, Enzo) and (B) trypan blue exclusion in cells transfected with Klf4 expression vector (300 ng/well) or empty vector (CT, control). * p < 0.05, ** p < 0.0001 vs. CT; (C) Analysis of caspase 3/7 activity. GCs transfected with Klf4 or empty vector were incubated without serum, and some control cells were treated with fetal bovine serum (FBS) or follicle-stimulating hormone (FSH) as positive controls for apoptosis suppressors. Twenty-four h later, caspase 3/7 activity was measured with the bioluminescence assay. FBS, 10% fetal bovine serum. * p < 0.05 vs. CT; (D) 5-bromo-2′deoxyuridine (BrdU)-incorporation assay. DNA labeling indices were determined by the scoring number of BrdU(+) nuclei per total nuclei, and are presented as means ± SDs. ** p < 0.001 vs. CT. Representative fluorescence microscopy images of BrdU-labeled GCs (100×) (lower panel). Cells were stained with BrdU antibody (B2531, Sigma, 1:200) to visualize BrdU-labeled cells (pink) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to confirm nuclear status (blue). CT, control cells transfected with empty vector; FSH, follicle-stimulating hormone (50 ng/mL); KLF4, cells transfected with Klf4 (300 ng).