Caveolin-1 Is a Critical Determinant of Autophagy, Metabolic Switching, and Oxidative Stress in Vascular Endothelium
Figure 4
Effects of caveolin-1 knockdown on endothelial mitochondria ROS metabolism, mitochondrial function and abundance.
(A) Analysis of mitochondrial ROS using the dye MitoSox Red in cells incubated in low-glucose DMEM medium (5.5 mM D-glucose) or after 6 hours in D-glucose-free DMEM medium containing 5.5 mM 2-deoxy-D-glucose (2DG). MitoSox fluorescence was analyzed by flow cytometry (n = 12). (B) BAEC were transfected with plasmids encoding mitochondria-targeted HyPer2 (HyPer-M) concomitantly with caveolin-1 or control siRNA. The cells were fixed 72 h after transfection and analyzed by HyPer2 ratiometric imaging and by immunofluorescence staining using the confocal microscope. Left, representative immunofluorescence, with caveolin-1 immunofluorescence signal in red and the HyPer2-M ratiometric image in green; the nuclear DAPI stain is in blue. The bar graph on the right shows the quantitative analysis of ratiometric signals pooled from analysis of 42 cells from three independent experiments. (C) and (D) Mitochondrial membrane potential assessed by labeling cells with the dye TMRM. Membrane potential was both quantitated by flow cytometry (panel C, n = 8) and analyzed by live cell imaging (D). (E) Co-localization of caveolin-1 with mitochondria in endothelial cells. Representative confocal images of endothelial cells transfected with caveolin-1 or control siRNA and subsequently stained with antibodies directed against caveolin-1 (cav-1, green) or the mitochondrial protein marker apoptosis inhibitor factor (AIF, red). Co-localization (yellow) is seen in the right-hand images, which also show nuclear staining with DAPI (blue). This experiment was repeated three times with similar results. (F) Representative immunoblots following siRNA-mediated caveolin-1 knockdown in endothelial cells. Immunoblots were probed with the mitochondrial marker protein cytochrome c oxidase subunit 4 (COX-IV), with β-actin as a loading control or with caveolin-1 to determine the extent of protein knockdown. (G) Mitochondrial abundance in control or caveolin-1 siRNA transfected cells assessed by flow cytometry using the probe MitoTracker Green FM (n = 4 each). All values are expressed as mean ± standard error of the mean (SEM). Statistical differences were assessed with unpaired t-tests: * indicates p<0.05, ** indicates p<0.01, NS, not significant.