Figure 6: Pacer LC3B imaging [FINAL]

Figure 6. pRAB7A-dependent function of Pacer in mitophagosome expansion. (A) Pacer KO HeLa cells stably expressing Parkin were transiently transfected with mCherry-Pacer WT and the pRAB7A binding mutant of Pacer characterized in Figure 4C-E. Cells were cotransfected with LAMP1-mNeon as a lysosomal marker and Halo-LC3 as an autophagosome marker. Cells were depolarized for 4 H in 10 μM Oligomycin A and 5 μM Antimycin A prior to live imaging and quantification in panels C, D, and E. Insets are 4 μm wide. (B) Pacer KO HeLa cells stably expressing Parkin and mCherry-WT Pacer/mCherry-Mutant Pacer were depolarized for 1 H in 10 μM CCCP. Cells were fixed, and imaged. Pacer puncta were counted using an automated particle counting program. 20 cells were analyzed and averaged in each biological replicate, and each cell’s puncta count was normalized to the average untreated puncta count for that day. 3-4 of these experiments were performed and averaged. Colored dots indicate technical replicates, black dots indicate averages for each experimental day, black horizontal line indicates averages of biological replicates. P-value calculated using a two-tailed T test of biological replicates. (C) Fold change in total autophagosome area. Cells imaged in panel A were masked and thresholded, and the total LC3 positive area was quantified. The total area for each cell was normalized to the average area for the mCherry transfected condition, and these values were averaged to calculate the value for a single biological replicate. This process was repeated in 4 independent experiments. P values were determined using Tukey’s multiple comparison test. (D) Fold change in LC3+/LAMP1+ positive area, determined as in panel C. (E) Fraction of total LC3 area that is also positive for LAMP1 as a metric for autophagosome-lysosome fusion efficiency, analysis performed as in panel C.