Figure 4

Quantification of CCCP induced mitophagy in HEK-293 and HCT-116 cells and deferiprone (DFP)-induced mitophagy in HeLa cells.

(A) Representative micrographs of HeLa cells transfected with Mito-Rosella and then treated with 1mM of 3-Hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone, DFP) or Vehicle (phosphate-buffered saline, PBS) for 24 hr. DFP treatment caused an accumulation of red-only Mito-Rosella labeled mitochondria (panel vi, solid arrows) while in vehicle treated cells there was complete co-localization between red/green signals (panel iii). Scale bar indicate a length of 50 μm. (B) Percentage of Mito-Rosella expressing cells showing increased mitophagy after 1 mM DFP treatment at indicated time-points as quantified by mito-Rosella FCM assay. Bars represent mean of 3 biological replicates ± SEM, N = 10,000 cells analyzed per replicate; *p < 0.001 vs DFP + BafA1 (C) Western blots of markers of mitochondrial content–HSP60, ATP synthase, VDAC1, TIM23 and TOM20 (top four panels) and p62 (autophagy marker) and actin (gel loading control) in cell treated with DFP or Vehicle (PBS) control for the indicated time-points. (D) Representative micrographs of HEK-293 cells transfected with Mito-Rosella alone (top panels) and HCT-116 cells (bottom panels) transfected with Mito-Rosella plus HA-Parkin; cells were treated with CCCP (10 μM) or vehicle as indicated; treatment durations were 24 hr for HEK-293 cells or 6 hr for HCT-116 cells. CCCP treatment induced accumulation of red-only Mito-Rosella indicating mitophagy (white arrows). Scale bars indicate a length of 25 μm. (E) Percentage of Mito-Rosella expressing HEK-293 and HCT-116 cells showing increased mitophagy after CCCP treatment as quantified by mito-Rosella assay.