Fig. 2
Effects of melatonin on high cholesterol-induced ROS accumulation and apoptosis. a–c UCB-MSCs were pretreated with DIDS (10 μM) prior to cholesterol treatment (200 μM, 24 h). a The intracellular ROS level was measured by CM-H2DCFDA. The relative fluorescence units (RFU) was measured by luminometer (n = 5). b The expression level of caspase 9 and 3 were quantified by western blotting (n = 5). c The percentage of apoptosis was analyzed with Annexin V/PI assay. Annexin V-FITC-positive and PI-positive (Q2), and Annexin V-FITC-positive and PI-negative (Q4) UCB-MSCs were considered as apoptotic (n = 5). d The cells were pretreated with melatonin (1 μM) or DIDS (10 μM) prior to cholesterol treatment (200 μM, 24 h), and the level of total ROS was measured by CM-H2DCFDA (n = 5). e The cells were pretreated with melatonin (1 μM) prior to cholesterol treatment (200 μM, 24 h) and then the expression levels of caspase 9 and 3 were measured by western blotting (n = 5). f The cells were pretreated with melatonin (1 μM) or DIDS (10 μM) prior to cholesterol treatment (200 μM, 24 h), and cell viability was measured with WST-1 assay (n = 5). All blot images are representative and quantitative data are presented as a mean ± standard error of the mean. *p < 0.05 vs control, #p < 0.05 vs cholesterol, $p < 0.05 vs melatonin + cholesterol, n.s. means not significant