Fig. 5: RhoA does not depolarize mitochondrial membrane potential or upregulate PINK1 mRNA but inhibits PINK1 protein degradation in cardiomyocytes.

A Mitochondrial membrane potential in NRVMs adenovirally infected with GFP (Ctrl) or RhoA was measured using TMRE, visualized by confocal microscopy, and cellular TMRE fluorescence intensity was quantified. Scale bars: 10 μm. n > 50 from three independent experiments; ns, not significant. B NRVMs were infected with GFP (Ctrl) or RhoA adenovirus for 16 and 24 h and PINK1 mRNA levels were assessed by qPCR. n = 6; ns, not significant. C GFP (Ctrl) or RhoA was co-expressed with msPINK1 for 16 h, and then treated with cycloheximide (CHX; 100 μg/ml) for the indicated times. Whole cell lysates were subjected to WB for PINK1 (msPINK1) and GAPDH (loading control). Arrows denote full length msPINK1, while lower bands are cleaved msPINK1. n = 5; *p < 0.05, ***p < 0.001. D GFP (Ctrl) or RhoA was co-expressed with msPINK1 for 16 h, and then treated with MG-132 (50 μM) for the indicated times. Whole cell lysates were subjected to WB for msPINK1 and GAPDH (loading control). Arrowheads denote cleaved msPINK1. n = 5, *p < 0.05, ***p < 0.001. E Cells expressing GFP (Ctrl) or RhoA were treated with MG-132 (50 μM) for the indicated times. Whole cell lysates were subjected to WB for PINK1 and GAPDH (loading control). Arrowheads denote cleaved endogenous PINK1. n = 4-5, **p < 0.01, ***p < 0.001.