Fig. 3

H/R-EMVs exhibited pro-apoptotic effect on H9c2 cells. a Hoechst 33258 staining was used to observe the apoptotic cells after treatment with 10, 30, 60 μg/mL H/R-EMVs for 6 h (100×). The arrows show apoptotic cells. The untreated cells serve as Control. b Annexin V-FITC/PI double staining. The results were interpreted in the following fashion: cells in the lower-left quadrant (Annexin V−/PI−) represent living cells; those in the lower-right quadrant (Annexin V+/PI−) represent early apoptotic cells; those in the upper-right quadrant (Annexin V+/PI+) represent late apoptotic cells; and those in the upper-left quadrant (Annexin V−/PI+) represent necrotic cells. c The apoptotic rate of H9c2 cells was quantified according to flow cytometry analysis. d Caspase 3 activity of H9c2 cells was measured after treatment with 10, 30, 60 μg/mL H/R-EMVs for 6 h. e Representative images of immunoblots with antibodies against Bcl-2, Bax, β-actin, respectively among groups. β-actin serves as a loading control. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus H/R-EMVs 10 μg/mL; ^& p < 0.05, ^&&& p < 0.001 versus H/R-EMVs 30 μg/mL