Fig. 3: RhoA activation increases mitophagy.

A–C Cells were infected with GFP (Ctrl) and RhoA adenovirus for 24 h and mitochondrial and cytosolic fractions were isolated for WB analysis. The lysosome inhibitor Bafilomycin A1 (BFA) was used at 50 nM. COX-IV and VDAC (mitochondrial markers) and Rho-GDI (cytosolic marker) were used as loading controls. n = 5–9, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. D, E Mitophagy was assessed by Mito-Keima. Mito-Keima was adenovirally co-expressed at 20 MOI in NRVMs with GFP (Ctrl) or RhoA for 32 h or treated with S1P at 1 μM for 1-2 h. LysoTracker Blue (250 nM) was loaded onto cells for 2 h prior to visualization. Merged images show all three colors and arrowheads to the purple dots indicate co-localization of Mito-Keima-560 nm fluorescence and LysoTracker Blue fluorescence signals. Scale bars: 10 μm. n > 40 from four independent experiments; *p < 0.05, ****p < 0.0001. F After 36 h infection of RhoA or GFP adenovirus in the absence or presence of BFA (50 nM), cells were collected and subjected to WB for VDAC, COX-IV, Lamin A/C and α-actinin, and band intensities were quantitated. n = 4–8, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.