Figure 4. Senescent cell derived sEVs confer anti-apoptotic activity. (A) Barchart of the top 20 most highly secreted sEV-miRNAs. To cell count normalized Ct-values from Q and SIPS from two time points were averaged and are plotted +/- SEM derived from all 12 samples. (B) Experimental setup to test the biological effect of the EV-SASP. Recipient fibroblasts were pre-exposed to sEVs for 24 hours followed by an acute stress treatment for 2 hours with 200 µM H2O2 and fresh sEVS were added. On the next day a second stress treatment with 400 µM for 2 hours was performed followed by a recovery time of 3 hours. Annexin-V-PI staining and flow cytometric measurement was used to determine % total number of apoptotic cells. (C) The EV-SASP reduces the amount of apoptotic cells of oxidatively stressed recipient cells. sEVs of SIPS and Q control cells of three different donors between 2 to 4 weeks of recovery post SIPS treatment were freshly harvested and applied before and after acute stress treatments. Human primary dermal (n = 3) and foreskin fibroblasts (n = 3) were used as recipient cells. Averages from 6 independent experiments +/- SEM are shown. Statistical analysis (n = 6) using 2-way RM ANOVA identified the factor 'EV/no EV' as a significant subject (p = 0.014) and the factor 'no stress/stress' as a significant factor (p = 0.00014). Groups were compared by Bonferroni post test, n.s ≥ 0.05; **p < 0.01, ***p < 0.01. (D) Representative pictures of recipient fibroblasts of all conditions tested prior Annexin-V-PI staining. Representative flow cytometric data are shown. Scale bar = 200 µm.