Fig 2: Proteomic analysis of human ES cells lacking FBXO7 during neurogenesis AWorkflow for analysis of total protein abundance in ES cells undergoing NGN2-driven neurogenesis with or without FBXO7. Cell extracts were digested with trypsin prior to 18-plex TMT labeling and analysis by mass spectrometry.BLog2 FC for the indicated mitochondrial protein-groups in control or FBXO7-/- cells at days 0, 4, or 12 during neurogenesis is shown in violin plots.CLog2 FC for the indicated cellular organelle proteins in control or FBXO7-/- cells at days 0, 4, or 12 during neurogenesis is shown in violin plots.DVolcano plots [Log2 FC (FBXO7-/-/Control) versus -Log10(q-value)] for FBXO7-/- (c52 and c89) and control cells at day 4 (left panel) or day 12 (right panel) of differentiation. Proteins showing decreased or increased abundance are shown as blue or red dots.ERelative abundance of DNM1L (Drp1), MFF, and FIS1 in control or FBXO7-/- cells at days 0, 4, or 12 during neurogenesis.F, GMitochondrial morphology in iNeurons (iN) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei, in either fed cells or cells treated with AO (3 h) (left panel). The number of median # of mitochondria/cell, median mitochondrial length, and median mitochondrial circularity is shown. Error bars depict SD from triplicates from three differentiations (9-12 image stacks each), as shown in panel I left, center, and right, respectively. Two-way ANOVA with multiple comparisons; *P = 0.0416, ****P < 0.0001. Scale bar = 10 µm.HWestern Blot analysis on HeLa control and FBXO7-/- whole cell lysate, probed for the mitochondrial fission adapter MiD51.IRelative abundance of DNM1L, MFF, MiD51, and FIS1 in WT (Control) or FBXO7-/- HeLa cells either in the fed state or after 16 h AO as determined from the proteomics data in Fig 5A and B.

Fig 3: Parkin and autophagy regulator recruitment in FBXO7-/- cells AOverview of methods for analysis of Parkin recruitment to damaged mitochondria.BCells were imaged for GFP-Parkin, nuclei, and mitochondria and segmented to facilitate analysis of Parkin recruitment to mitochondria in single cells/mitochondrial organelles.CStd mitochondrial signal vs. Parkin intensity and Std Parkin vs. mitochondria with or without 1 h AO-induced mitophagy are shown. Data from three replicates, including a total of 238 image stacks, containing 16,888 cells and 237,848 mitochondrial objects.DRecruitment of p62 to mitochondria (not stained) in control or FBXO7-/- HeLa cells with or without treatment with AO (16 h) was examined by confocal imaging. Scale bar = 10 and 5 µm.E, FQuantification of cells in panel (D). Assays were performed in biological triplicate with 4 image stacks taken per repeat. n = 6,712 cells. Error bars depict SD from triplicate experiments from two independent clones. One-way ANOVA with multiple comparisons; ***P = 0.0002, *P = 0.0355, **P = 0.0029, ****P < 0.0001.GQuantification of number of p62 foci normalized per cell in control, PINK1-/- and FBXO7-/- cells with or without 3 h AO treatment. Assays were performed in biological triplicate with 4 image stacks taken per repeat. n = 12,646 cells. Error bars depict SD from triplicate experiments from two independent clones. One-way ANOVA with multiple comparisons; ****P < 0.0001.HControl or FBXO7-/- HeLa cells expressing Parkin were either left untreated or incubated with AO for 1 or 6 h and extracts subjected to Western Blotting with a-LC3B and a-Actin as a loading control.IThe ratio of LC3B lipidation (LC3B-II/LC3B-I) was quantified from triplicate experiments from three independent clones. One-way ANOVA with multiple comparisons. *P = 0.0104.JImmunofluorescence staining of WIPI2 (magenta) and Tom20 (gray) in HeLa Control and FBXO7-/- cells after mitophagy induction. Arrows depict WIPI2 foci on the mitochondrial staining. Scale bar = 10 µm (overview) and 10 µm (insets).KEvaluation of mitochondrially localized WIPI2 foci per cell based on images shown in (J). Data based on 15 image stacks from three technical replicates of two independent clones. One-way ANOVA with multiple comparisons. *P = 0.0331.LImmunofluorescence staining of HEK293T Control and FBXO7-/- cells expressing GFP-Parkin for HSP60 (mitochondria, magenta), pUb (gray) and GFP (Parkin, green) after mitophagy induction for indicated times. Scale bar = 10 µm. Number of individual cells analyzed per condition is indicted in the right bottom of each image.MAnalysis of pUb intensity (left) and Parkin intensity (right) in the mitochondrial mask. Error bars depict SEM from three biological replicates from two independent clones. 2-way ANOVA with multiple comparisons; **P = 0.001, ****P < 0.0001.

Fig 4: Mitophagic flux in iNeurons and HeLa cells lacking FBXO7 AScheme depicting mitophagic flux assay using mtKeima. See text for details.BMean Acidic:Neutral mtKeima per-cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO (Antimycin A [5 µM] and Oligomycin [10 µM]); or 3 h with 25 nM BafilomycinA (BafA). Error bars depict SD from biological triplicate measurements from three independent clones. Two-way ANOVA with multiple comparisons; ****P < 0.0001.CBar graph showing the number of mtDNA puncta/cell with or without treatment with AO (6 h) in HeLa control or FBXO7-/- cells. Error bars depict SD from triplicate technical replicates from two independent clones. One-way ANOVA with multiple comparisons; ****P < 0.0001.DIndicated hESCs expressing mtKeima were differentiated to iNeurons (day 12) and were subjected to image analysis. Scale bar, 5 µm.EThe number of red-shifted Keima foci per cell is plotted, where each dot represents the average foci-number per image stack, originating from 7 to 12 image stacks of iNeuron differentiations. Error bars represent SD. One-way ANOVA with multiple comparisons; ***P = 0.0001, ****P < 0.0001.FmtKeima mitophagy flux readout of indicated iNeuron genotypes. Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry (> 10,000 cells). Pooled data from biological replicates is shown, normalized to 6 h BafA treated cells.GBar graph showing the number of mtDNA puncta/cell with or without treatment with AO (6 h) in control or FBXO7-/- day 12 iNeurons. Error bars depict SD from biological triplicate measurements from two independent clones and three differentiations. One-way ANOVA with multiple comparisons.

Fig 5: Super-resolution pUb detection in HeLa cells in response to mitochondrial depolarization3D-SIM images of HeLa control, PINK1-/- and FBXO7-/- cell lines after 1 h, 3 h, or 6 h, AO-induced mitophagy (related to Fig 2A and B). Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Note that 1 h AO panel is reused from Fig 2A for side-by-side comparison. Scale bar = 5 µm or 1 µm.