Fig. 3: Pptc7 knockout causes excessive BNIP3- and NIX-mediated mitophagy.

a Western blot of BNIP3 and NIX expression in wild-type, Pptc7 KO, and two Pptc7/Bnip3/Bnip3l triple knockout (TKO) MEF cell lines. Actin is shown as a load control. This experiment is representative of at least three independent experiments. b Representative images of wild-type, Pptc7 KO, and each TKO cell line expressing the mitophagy reporter mt-Keima. Mitochondria imaged at 458 nm are at physiological pH but those imaged at 561 nm reflect acidic mitochondria undergoing mitophagy. c Quantification of mt-Keima imaging. Each gray dot represents the ratio of mitochondrial fluorescence from a single cell; n = 123 for wild type, n = 152 for Pptc7 KO, n = 123 for TKO#1, n = 143 for TKO#2. Blue, orange, and purple dots represent averages from three independent biological experiments. The bar graphs intersect the mean of these three experiments; error bars represent standard deviation. ****p < 0.0001. mt-Keima microscopy data was analyzed by ordinary one-way ANOVA. d Quantification of mt-Keima as analyzed by FACS. Blue, orange, and purple dots represent averages from three independent biological replicates. The bar graphs intersect the mean of replicates; error bars represent standard deviation. mt-Keima microscopy data was analyzed by a Brown-Forsythe and Welch ANOVA. *p < 0.05, ns = not significant. For WT v. KO, p = 0.0341, for KO v. TKO #1, p = 0.0428, for KO v. TKO #2, p = 0.0332. e Proteomic analysis of mitochondrial proteins in Pptc7 knockout relative to wildtype (green), Bnip3 knockout relative to Pptc7 knockout (pink), Nix knockout relative to Pptc7 knockout (orange), and two independent Pptc7/Bnip3/Bnip3l TKO lines normalized to Pptc7 KO (dark blue and light blue). Each dot represents a quantified mitochondrial protein; n of each experiment is reported below the x-axis. Proteomic data was analyzed by ordinary one-way ANOVA. ****p < 0.0001. Mean and standard deviation shown. f Seahorse analysis of a mitochondrial stress test in immortalized wild type (WT, black), Pptc7 KO (red), TKO #1 (dark blue), TKO #2 (teal) were assayed in Seahorse DMEM supplemented with 25 mM glucose. Data are represented as the mean of n = 22 replicate wells per condition; error bars represent standard deviation. g Seahorse analysis of basal oxygen consumption rates (OCR) and extracellular acidification (ECAR). Mean of n = 8 independent wells shown; error bars represent standard deviation. h, i BNIP3 (h) and NIX (i) protein expression across 207 cell lines harboring monogenic mutations in genes encoding mitochondria-localized proteins. Data were analyzed via a two-sided Student’s t test with log-transformed p-values reported on the y-axis. Only Pptc7 KO increases both BNIP3 and NIX significantly across this dataset. Source data are provided as a Source data file.