Figure 2

Single-cell measurements of autophagy and mitophagy based on biosensor responses to CCCP treatment.

(A) Representative micrographs of HeLa cells transiently co-transfected with Rosella-LC3 expression plasmid plus Parkin expression plasmid or ‘empty’ plasmid (Control). Cells were treated for 24 hr with: Vehicle alone (DMSO), CCCP (10 μM), or CCCP (10 μM) + lysosomal inhibitor Bafilomycin-A1 (BafA1, 100 nM). CCCP-treated cells displayed accumulation of primarily red Rosella-LC3 fluorescence near the central lysosomal compartments (solid arrows in identical, labeled panels below panels vi and xv), while vehicle treated cells primarily display co-localization between red and green signals (hollow arrows in identical, labeled panels below panels iii and xii). Co-treatment with BafA1 (panels ix and xviii) prevented CCCP-induced relocation of Rosella-LC3 to the central lysosomal compartment, causing it to accumulate in autophagasomes within the cytoplasm (panels ix and xviii). (B) Representative micrographs of HeLa cells co-transfected with Mito-Rosella plus either Parkin or Control plasmids and treated exactly as described above for (A). CCCP treatment caused an accumulation of red-only Mito-Rosella-labeled mitochondria in the central lysosomal compartments in Parkin co-transfected cells but not in control cells (solid arrows in identical, labeled panel below panel xv) and this was inhibited by BafA1 treatment (panel xviii). In contrast, DMSO-treated Parkin-expressing cells (panel xii) and control cells treated with vehicle (DMSO) or CCCP (panels iii and vi), there is a complete co-localization of green/red fluorescence. Quantification of percentage of cells exhibiting induction of (C) autophagy or (D) mitophagy, as determined by categorizing individual cells on the basis of presence/absence of visible accumulations of red biosensor fluorescence in the lysosomal compartments as in (A) and (B) above. For panels C and D: bars represent mean ± SEM of percentages of cells from n = 4 biological replicates, with >120 cells categorized per replicate. For panels A and B: scale bars indicate a length of 25 μm. For panel C: *p <  0.05 vs. DMSO treated cells at the same time-point; and ^#p  <  0.05 vs. 6 hr time-point by unpaired T-test. For panel D: *p <  0.05 by unpaired T-test for the indicated comparisons between cells co-transfected with Parkin vs. Control plasmid.