Figure 2.

Exosomes released from hypoxia-pretreated CMs protect CMVECs from oxidative stress injury. CMVECs were pretreated with CM-exos (300 μg/ml) for 24 h before incubation with 200 μM H₂O₂ for 3 h and then subjected to analysis. (a) Representative dot plots of cell apoptosis after Annexin V/PI dual staining are shown. The left upper quadrant (% gated) shows necrotic cells (Annexin V−/PI+); the upper right quadrant (% gated) shows late apoptotic cells (Annexin V+/PI+); the lower left quadrant (% gated) shows live cells (Annexin V−/PI−); and the lower right quadrant (% gated) shows early apoptotic cells (Annexin V+/PI−). (b) The percentage of apoptotic cells represents total apoptotic cells, including both early and late apoptotic cells; n = 3. (c) Representative dot plots of cell apoptosis after Annexin V/PI dual staining are shown. (d) The percentage of apoptotic cells represents both early and late apoptotic cells; n = 3. (e) The intracellular ROS level was determined by FCM. The P2 percentage indicates the proportion of cells with increased ROS production, with signals above background 2′,7′-dichlorofluorescein (DCF) fluorescence levels. (f) Quantitative analysis of ROS levels; n = 3. (g) Graph represents SOD levels; n = 9. (h) Graph represents MDA levels; n = 9. (i) Representative immunofluorescence of TUNEL (green) and DAPI (blue) staining and merged images. Photos were randomly captured using a fluorescence microscope. Scale bar = 20 μm. (j) The panel shows the percentage of TUNEL-positive cells; n = 6. (k) Apoptosis-related genes, such as procaspase-3, cleaved caspase-3, Bax, and Bcl-2, were detected by immunoblotting. (l) Quantitative analysis of the apoptosis-related proteins; n = 3. ^∗P < 0.05 compared with the control group; ^#P < 0.05 compared with the H₂O₂ group; ^&P < 0.05 compared with the Nor-exos group.