Fig 2: DPD induction of mitophagy in GBM cells A, C) ROS (green fluorescence) expression after treatment of U87MG and LN229MG cells with 0, 15, and 30 µM DPD for 48 h. Confocal microscopy images (A), scale bar = 20 μm. Flow cytometry was used to assess ROS levels (B), and a histogram analysis was performed (C). D, F) Confocal microscope images of JC-1 staining in U87MG and LN229MG cells after treatment with 0, 15, and 30 µM DPD, and CCCP for 48 h (D), scale bar = 20 μm. Flow cytometry quantitative analysis (E) and histogram analysis (F). G) ATP concentrations after DPD treatment of GBM cells. H) BNIP3L, Bcl-2, Beclin-1, and TOM20 protein expression after DPD treatment of GBM cells. I) BNIP3L, LC3, and p62 protein expression after 3-MA and/or DPD treatment of GBM cells. J) Co-localized visualization of BNIP3L and Bcl-2 by laser confocal microscopy, scale bar = 10 μm. (*P < 0.05; ***P < 0.001; ****P < 0.0001, ####P < 0.0001, compared with control)

Fig 3: PPTC7 interacts with and can dephosphorylate BNIP3 and NIX.a Mitochondrial proteins that interact with PPTC7 after affinity purification-mass spectrometry analysis as performed in ref. 22. b Immunoprecipitation (IP) of FLAG-PPTC7 overexpressed in U2OS cells (right panels) with endogenous BNIP3 and NIX in basal conditions (middle column) or after treatment with 1 mM DFP to induce BNIP3 and NIX expression (right column). Input shown as whole cell lysate (WCL) at right; anti-vinculin (VCL) shown as a load control. This experiment is representative of at least three independent experiments. c Yeast two-hybrid analysis of PPTC7, BNIP3, and NIX as reciprocal baits and preys. Yeast were spotted on permissive (+his, left) or selective (-his, right) plates. d, e Phosphoproteomic analysis of BNIP3 (d) or NIX (e) phosphopeptides isolated from Pptc7 knockout mitochondrial fractions. Mitochondria were isolated from cells and split into n = 3 fractions for each treatment: untreated (black dots, left, n = 3 mitochondrial fractions), treated with catalytically inactive PPTC7 (D78A, blue dots, middle, n = 3 mitochondrial fractions) or active PPTC7 (orange dots, right, n = 3 mitochondrial fractions). The log2 intensity of each phosphopeptide is shown; each dot represents a biological replicate from one of the nine independently treated mitochondrial fractions outlined above. Lines represent median. Ordinary one-way ANOVA performed. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant. p-values for WT v. D78A are as follows: BNIP3 S60 p = 0.001, BNIP3 T66 p = 0.1145, BNIP3 S79 p < 0.0001, BNIP3 S88 p < 0.0001, NIX S63,64 p = 0.0003, NIX S64 p < 0.0001, NIX S119 p < 0.0001, NIX S165 p = 0.0001. Source data are provided as a Source data file.

Fig 4: 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.

Fig 5: Knockdown of a BORC subunit recapitulates the phenotypes of Y102 treatment. (A) A western blot of lysates from U87-MG cells treated for 72 h with negative control siRNA (siNeg) or siRNA against BORCS6 (siBORCS6) was probed for BORCS6. Densitometry analysis was used to determine the ratio of BORCS6 to α-Tubulin control to determine sufficient knockdown. Densitometry analysis was used to determine their ratio for the blot shown. Blot is representative of two experiments. (B) U87-MG cells were treated with negative control siRNA (siNegative) or siBORCS6. Following 72 h knockdown, cells were stained for the lysosome marker LAMP1 (red), the mitophagy receptor BNIP3L (green), and the autophagy marker p62 (red). DAPI was used to counterstain cell nuclei (blue). Scale bars: 150 µm. Images are representative of three repeats. (C) U87-MG cells were treated for 72 h with siNeg or siBORCS6. At 3 h prior to collection, cells were stained with alamarBlue. Results are the mean±s.d. (n=3). ***P<0.0003 (unpaired two-tail t-test).