Figure 6. Analysis of PM-induced senescence in ovarian somatic cells. (A–C) Cells treated with 10 μM PM for the indicated time were analyzed by flow cytometry in order to quantify (C) cell cycle distribution and (B) apoptosis (sub-G1). Results are expressed as mean ± SEM of three experiments. Statistical differences vs control **p<0.01 ****p<0.0001. Statistical differences vs 16hrs in the G2/M phase a = p<0.05 c = p<0.001 d = p<0.0001. (D, E) Comparison of qRT-PCR for (d) p21 and (e) ki67 between in vitro cultured dispersed ovarian cells and ovarian fragments. Data are shown as mean ± SEM of three analyses. Statistical differences vs control *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. (F, G) Representative staining for Click-iT EdU (green) in (F) scattered cells and in (G) little colony (white circle) after 72 hrs of treatment with PM. Red arrowheads indicate proliferating cells. Scale bar = 100μm. (H) Cultured ovarian somatic cells acquired large and flattened morphology from 72 hrs of culture with PM. (I, J) Representative image and quantification of cells positive for SA-βgal activity after 120h of PM treatment. Data are expressed as mean ± SEM of SA-βgal positive cells percentage scored in three experiments. Statistical differences vs control **p<0.01 ***p<0.001. Scale bar=100μm.