Figure 3

Parallel quantification of autophagy and mitophagy under the same conditions by flow cytometry (FCM) measurement of biosensor responses.

Representative histograms of pHluorin/green and DsRed.T3/red fluorescence intensities of HeLa cells transiently transfected with plasmids expressing (A) Rosella-LC3 and Parkin, or (B) Mito-Rosella and Parkin and treated for 24 hr with: DMSO (vehicle), CCCP (10 μM), or CCCP (10 μM) + Baf1A (100 nM); histograms depict intensities of n = 10,000 cells per condition. CCCP induced a significant reduction (dashed lines and arrow) in pHluorin/green fluorescence but not in DsRed.T3/red fluorescence of both Rosella-LC3 and Mito-Rosella. Quantitation of cells with (C) increased autophagy or (D) increased mitophagy based on detection of changes in green vs. red fluorescence intensities in gated FCM scattergram analysis (see also Suppl. Fig. 5A–B); bars represent means of 3 biological replicates ± SEM, N = 10,000 cells analyzed per replicate; *p < 0.001 vs. CCCP + BafA1; #p < 0.001 vs. DMSO at 6 time-point (see also Suppl. Fig. 5A–B). (E) Western blots of LC3 processing in cells co-transfected with Rosella biosensors plus Parkin or Control plasmids and treated 24 hr as indicated. (F) Western blots of mitochondrial markers in cells co-transfected with plasmids for each of the Rosella biosensors plus Parkin or Control plasmid. (G) Representative micrographs of HeLa cells stably expressing Mito-Rosella and transiently transfected with a bicistronic iRFP-T2A-Parkin expression plasmid; iRFP fluorescence was readily visualized and exhibited a diffuse localization in successfully transfected cells (panels iv and ix). As expected, DMSO did not induce mitophagy in cells expressing Parkin (iRFP positive cells; panels iii–v). Consistent with our other findings, CCCP treatment did not induce mitophagy in iRFP-negative cells lacking Parkin (panels vi–x, solid white arrow), but induced mitophagy in iRFP-positive cells expressing Parkin (panels viii–x). (H) FCM analysis was used to demonstrate the requirement of Parkin expression for CCCP-induced mitophagy. Cells stably expressing Mito-Rosella and transiently transfected with iRFP-T2A-Pakin were first gated according to negative vs. positive iRFP-fluorescence (i.e., No Parkin vs. Parkin expression, respectively) and then analyzed separately to detect mitophagic cells (see Suppl. Fig. 5D), using the same method described for Fig. 3C,D.