Figure 1

RSV inhibited t-BHP-induced injury in HUVECs. (a and b) Cells were treated with t-BHP with different concentrations (20, 30, 40, 50, 60, 70, 80, 90 and 100 μM) for 4 h (a) or for different time intervals (1, 2, 4, 8, 12 and 24 h; b), respectively. Cell viability was determined using the CCK-8 assay and data are expressed as percentage of the control. (c) Confluent cells were pretreated for 2 h with various concentrations of RSV (0.1, 0.5, 1, 5, 10 and 15 μM). After removing the supernatants, cells were incubated with fresh medium in the presence or absence of t-BHP (80 μM) for an additional 4 h. Cell viability was determined using the CCK-8 assay. (d) Representative images of flow cytometric analysis by annexin V-FITC/PI dual staining. The bottom right quadrant represents annexin V-FITC-stained cells (early-phase apoptotic cells) and the top right quadrant represents PI- and annexin V-FITC-dual-stained cells (late-phase apoptotic/necrotic cells). (e) Apoptotic cells are represented as the percentage of annexin-V single-positive plus annexin-V/PI double-positive cells. (f) After the indicated treatments, the cells were harvested and lysed to detect the cytoplasmic and mitochondrial levels of Bax and Bcl-2 by western blot analysis. (g) The bar graphs show the ratio of Bax/Bcl-2 in the cytosolic fraction in endothelial cells. (h) The bar graphs show the ratio of Bax/Bcl-2 in the mitochondrial fraction in endothelial cells. (i) The expressions of AIF and CytC in the cytosolic fraction of endothelial cells were detected. (j) The bar graphs show the quantification of the indicated proteins. All results are representative of three independent experiments and values are presented as means±S.E.M. (n=3). ^aP<0.05, ^bP<0.01 versus the control group; ^cP<0.05, ^dP<0.01 versus the t-BHP-treated group