Fig. 1.

Caveolin-1 Regulates Mitochondrial Shape and Susceptibility to Damage. MDA-MB-231 cells treated with control siRNA (CTL) or Cav-1 siRNA (SI) were stained with 50 nM TMRM for 30 min or 5 μM MitoSox for 10 min at 37 °C and visualized by confocal microscopy.

(A) Over 30,000 individual mitochondria were analyzed with an ImageJ plugin and categorized into different length groups. Quantification revealed that subpopulations with the shortest and longest length both increased, whereas the number of mitochondria in the intermediate group decreased in Cav-1 depleted cells.

(B) Average length was calculated by dividing total length by the number of mitochondria. Mitochondrial length after Cav-1 knockdown was reduced by 15% compared to control. Data are mean ± SEM, n ≥ 15; *p < 0.05 vs control by t-test.

(C) Mitochondrial ROS was measured by fluorescence intensity of MitoSox. Depletion of Cav-1 reduced mitochondrial ROS by 26% as compared to control siRNA group. Data are mean ± SEM, n = 10; ***p < 0.001 vs control by t-test.

(D) Cells infected by retrovirus encoding mito-roGFP2-ORP1 were treated with 10 μM doxorubicin for 4 h and then visualized by confocal microscopy. The fluorescence ratio at 510 nm peak emission from excitation at 405 nm and 488 nm reflects mitochondrial oxidative status. Index of relative oxidation (I_RO) was calculated using equation I_RO = 1-(R-R_H2O2)/(R_DTT- R_H2O2). Cells with low Cav-1 expression exhibited reduced level of mitochondrial oxidation even after challenged by doxorubicin. Data are mean ± SEM, n ≥ 10; *p < 0.05, ***p < 0.001 by ANOVA.

(E) After treating with 10 μM doxorubicin for 24 h, cytosolic fractions were collected and analyzed by RT-PCR. Bar graph summarizes the fold change of mtDNA in the cytosol normalized to control. Cav-1 inhibition diminished mitochondrial damage induced by doxorubicin by over 50%. Data are mean ± SEM, n = 3–5; *p < 0.05, **p < 0.01 vs control by ANOVA.