Research Paper Volume 11, Issue 21 pp 9912—9931

Molecular basis of senescence transmitting in the population of human endometrial stromal cells

Figure 4. Soluble factors and extracellular vesicles secreted by senescent ESCs trigger senescence in young cells. Sen – senescent ESCs. Ctr – young ESCs cultured in standard conditions. SF-sen or EV-sen treated – young ESCs exposed to soluble factors and extracellular vesicles secreted by senescent ESCs, respectively. (A) Western blot analysis of CD63 and HSP70 total proteins amount in Sen and EV-sen lysates. (B) and (C) Growth curves and cell size of Ctr, SF-sen and EV-sen treated ESCs determined by FACS. Forward scatter (FS) reflects the average cell size evaluated after 6 d of exposure. Values are M ± S.D. (N=3). *** – p<0.005 by ANOVA with Tukey HSD versus Ctr. (D) Western blot analysis of p53 and Rb phosphorylation levels and p21 protein expression performed after 7 d of treatment. Representative results of the three experiments are shown in the Figure. GAPDH was used as loading control. (E) SA-β-Gal staining of Ctr, SF-sen and EV-sen treated ESCs. After 7 d of treatment ESCs were reseeded and additionally cultured for 3 d in order to perform staining of non-confluent cultures. (H) Quantification of SA-β-Gal activity values (E). Values presented as M and 95 % CI (N=100). *** – p<0.005 by ANOVA with Tukey HSD versus Ctr.