Figure 4. smARF induces morphological indicators of mitophagy. (A) Electron microscopy of U2OS-smARF cells untreated or treated with doxycycline for 48 h (0.1 µg/mL). The arrowheads indicate swollen mitochondria evident after smARF induction. The data depicted are representative of several independent experiments, and two different subclones of U2OS-smARF cells. Scale bar: 500 nm. (B) U2OS-smARF cells were treated with either doxycycline (doxy, 0.1 µg/mL) or a positive control (the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone, CCCP, 10 μM) for 48 h or left untreated (unt). The mitochondrial transmembrane potential was measured using the flow cytometry-based MitoPotential assay from Millipore (Guava EasyCyte). Note that induction of smARF increases the percentage of cells with depolarized mitochondria. The graph depicts data from three independent experiments, with standard error. (C) U2OS-ARF cells were transiently transfected with GFP-LC3 plasmid for 24 h, treated with doxycycline for 48 h (0.1 µg/mL) and examined by confocal microscopy. The chart shows the percentage of cells containing more than four GFP-LC3 vacuoles compared with untreated controls. The results are the average plus standard error of three independent experiments in which at least 200 cells were counted for each sample. (D) Immunofluorescence analysis of U2OS-smARF cells transiently transfected with GFP-LC3 plasmid for 24 h, then untreated or treated with doxycycline for 48 h (0.1 µg/mL) and stained with MitoTracker Red. The bottom panels depict the colocalization of GFP-LC3 vacuoles with MitoTracker stain following smARF induction. (E) U2OS cells with inducible smARF were left untreated or incubated with 0.1 µg/mL doxycycline for 48 h, and mitochondrial mass was assessed following incubation with MitoTracker Green (MTG). The data depicted are representative of three independent experiments. (F) U2OS cells with inducible smARF were left untreated or incubated with 0.1 µg/mL doxycycline for 24 h, and then analyzed by immunofluorescence for TOMM20. The data depicted are representative of three independent experiments.