Figure 5. Effects of CCCP vs H₂O₂ on Rab5 recruitment to mitochondria.

BAC GFP-Rab5 HeLa cells labeled with 100 nM of Mito-Red were treated with either DMSO (Ctrl), 10 μM CCCP (A), or 250 μM H₂O₂ at 37°C for 2 hr (B). Cells were fixed and imaged by confocal microscopy. Inset regions reveal the effect on mitochondrial morphology and GFP-Rab5 localization upon treatment. Arrowheads indicate rounded and stressed mitochondria in both CCCP- and H₂O₂- treated conditions. Scale bars, 10 μm. (C) and (D) Colocalization analysis between Mito-Red and GFP-Rab5 in (A) and (B), respectively; n = 50. Error bars represent SEMs. p Values based on two-tailed t-tests. (E) Subcellular fractionation was performed in HeLa cells treated with DMSO (Ctrl), 10 μM CCCP, or 250 μM H₂O₂ at 37°C for 2 hr. Protein samples from purified mitochondria (Mito) and cytosolic (C) fractions were loaded onto SDS-PAGE and imunoblotted with cytochrome c antibody. (F) Cells were treated the same way as in (E). Cells were then resuspended in live-cell imaging solution containing 500 nM caspase-3/7 green flow cytometric reagent and incubated at 37°C for 30 min before subjecting to flow cytometric analysis. FITC signal (x-axis) is plotted against the total cell count (y-axis). The gating was set based on the background signal in DMSO control. (G) HeLa cells were seeded in the Seahorse 96-well plate format and incubated overnight. Growth medium was exchanged to bicarbonated-free low-buffered assay medium (provided by the manufacturer) supplemented with 10 mM galactose immediately before the start of the experiment. Oxygen consumption rate measurements were measured in the Seahorse XFe96 Analyzer from cells injected with media only (purple), 250 µM H₂O₂ (green), or 10 µM CCCP (orange). Error bars represent SEMs.

Figure 5—figure supplement 1. CCCP, but not H₂O₂, leads to Parkin recruitment.

(A) BAC GFP-Parkin or WT HeLa cells (B) were treated with either DMSO (Ctrl), 10 µM CCCP, or 250 µM H₂O₂ at 37°C for 2 hr. Cells were fixed and imaged by confocal microscopy. HeLa cells were immunostained with Parkin antibody. Inset regions are shown. Scale bars, 10 μm.