Fig. 3

Enhanced oxidative stress and apoptosis in aging granulosa cells. A Senescent KGN were identified through SA-β-galactosidase (SA-β-gal) staining following treatment with H₂O₂ (300 µM, 4 h). B Evaluation of relative mRNA expression levels for genes associated with cellular senescence and the senescence-associated secretory phenotype (SASP) (n = 3 for each group). C Intracellular reactive oxygen species (ROS) levels were assessed after H₂O₂ treatment. Representative fluorescence images depicting aging KGN. D Quantitative analysis of DCFDA staining results (n = 3 for each group). E Measurement of mitochondrial membrane potential (MMP) by JC-10 staining after H₂O₂ treatment. Representative fluorescence images displaying aging KGN. JC-10 aggregates emit orange-red fluorescence while JC-10 monomers emit green fluorescence. F Flow cytometric assessment of MMP using flow cytometry and presentation of statistical data for MMP analysis (n = 4 for each group). G Annexin V and propidium iodide (PI) staining in KGN treated with H₂O₂ (300 µM, 4 h). And statistical representation of the apoptosis rate in senescent KGN (n = 4 for each group). Ctrl: control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001