Figure 5: Pacer Flux [FINAL]

Figure 5. Pacer is a pS72 RAB7A-dependent activator of mitophagy. (A) Su9-HaloTag processing assay conducted as in Figure 1C. Parkin-expressing cells were treated with either complete medium, or complete medium supplemented with 10 μM Oligomycin A, 5 μM Antimycin A for 4 H. Each lane represents lysates from separate experiments (B) Quantification of panel A, conducted as in Figure 1D. n = 3 for the uninduced condition, n = 9 for the induced conditions. Error bars indicate standard deviation, and the p-value was calculated using a one- tailed T test. (C) Pacer KO cells expressing Parkin and Su9-HaloTag reporter were stably transfected with either Pacer, the pRAB7A binding mutant Pacer characterized in Figure 4C-E, or empty vector, and then depolarized for 2 or 4 H. n = 3 independent experiments. Pacer expression was probed via Western blot, while HaloTag processing was directly imaged using a fluorescent Halo ligand. (D) Quantification of panel C. P-value was calculated using a one-factor ANOVA at 2 and 4 H. (E) The same cells used in panel C were exposed to the non-Parkin mitophagy inducer DFP for 8, 16, and 24 H, and analyzed as with previous Su9-Halo processing blots. (F) Quantification of panel D. n = 4 independent experiments. P-value was calculated using a one factor ANOVA at each timepoint. (G) Pacer KO cells expressing LC3-Halo were stably transfected using either WT Pacer, pRAB7A-binding mutant Pacer, or empty vector, and the starvation autophagy flux assay was conducted as in Figure 1a. (H) Quantification of panel F. Between 3 and 9 independent experiments were conducted for each timepoint. P value was calculated using a one-factor ANOVA at each timepoint.