Caveolin-1 Is a Critical Determinant of Autophagy, Metabolic Switching, and Oxidative Stress in Vascular Endothelium
Figure 1
Effects of siRNA-mediated caveolin-1 knockdown on ROS levels in cultured endothelial cells.
BAEC were transfected with a caveolin-1 or control siRNA, and analyzed 72 h following transfection. (A) Amplex Red assay for H2O2, pooled data from six independent experiments, each performed in duplicate. (B) Flow cytometry experiments measuring intracellular ROS levels using CM-H2DCFDA in control and caveolin-1 siRNA-transfected BAEC (n = 12). (C) Ratio of reduced to oxidized glutathione (GSH/GSSG ratio) in control and caveolin-1 siRNA-transfected cells (n = 8 each). (D) Intracellular H2O2 levels assessed by HyPer2 ratiometric analysis of BAEC co-transfected with caveolin-1 siRNA and Hyper2 plasmid DNA; 72 h later cells were fixed and analyzed by fluorescent microscopy; separate coverslips were analyzed for caveolin-1 immunofluorescence. Left, photomicrographs shows representative immunofluorescence images, with the caveolin-1 signal in red, and the DAPI nuclear stain in blue. Right, representative ratiometric images showing HyPer2 in control and caveolin siRNA-transfected cells; the bar graph on the far right shows pooled data from 48-50 cells, presenting quantitative analysis of the ratiometric intensity analyzed in three separate experiments. For this experiment, addition of H2O2 (100 µM) serves as a positive control for the HyPer2 biosensor. (E) Catalase activity measured in control and caveolin-1 siRNA transfected cells in the presence and absence of the inhibitor 3-amino-1,2,4-triazole (ATZ). All values are expressed as mean ± standard error of the mean (SEM). Statistical difference was assessed with unpaired t-test; * indicates p<0.05.