Fig. 2.

Depletion of Cav-1 Increases Mitochondrial Dynamics. MDA-MB-231 cells treated with control siRNA (CTL), Cav-1 siRNA (SI), or Cav-1 adenovirus (AD) to rescue Cav-1 expression for 48–72 h were stained with 50 nM TMRM for 30 min at 37 °C and visualized by confocal microscopy.

(A) Mitochondrial fusion and fission events per minute were increased in Cav-1 depleted cells (SI) as compared to control and Ad-Cav-1 rescue group.

(B) Mean speed of mitochondrial movement was quantified by Imaris software. The motility of mitochondria in Cav-1 depleted cells increased significantly. Data are mean ± SEM, n = 15; *p < 0.05, **p < 0.01, ***p < 0.001 vs control by ANOVA.

(C) CTL, SI and AD cells were co-transfected with mito-Ds-Red and mito-Photoactive (PA)-GFP for 48 h. Live cells were visualized by confocal microscopy. Mitochondrial Networking Factor (MNF) which represents the area of photoactivated GFP spreading was calculated using ImageJ. The loss of Cav-1 enhanced MNF demonstrating wider spreading of activated GFP which was inhibited by rescuing Cav-1 expression.

(D) CTL, SI and AD cells were transfected with mito-Ds-Red for 48 h. Live cells were visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) performed on cells after Cav-1 knockdown ± Ad-Cav-1 rescue were transfected with mito-DS-Red. Mean fluorescence recovery curves after photobleaching region of interest (ROI) normalized to the last prebleach image.

(E) Quantification of fluorescence recovery rate revealed greater fluorescent signal recovery in Cav-1 depleted cells.

(F) Quantification of recovery half time revealed faster fluorescent signal recovery in Cav-1 depleted cells. Data are mean ± SEM, n ≥ 10; *p < 0.05, **p < 0.01, ***p < 0.001 vs control by ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)