mouse bnip3  (Vector Biolabs)

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A) New born tibia from MitoQC mice show mitophagy (red punctae) in articular chondrocytes. (B) New born tibia from MitoQC mice show mitophagy (red punctae) in osteoblasts (near Bone lining (BL) and Perichondrium (PL) and osteocytes (Ocy). The different panels show DAPI stained nuclei, EGFP and MCherry indicate mitochondria and the overlay show red only punctae indicating mitophagy. (C) Calvarial osteoblasts (COB) from MitoQC mice treated with VEH (methanol, top pane) and DFP (1mM, bottom row). The red punctae increased with DFP treatment, indicating increased mitophagy. Representative confocal images from n=3 samples. (D) Quantification of Red/Green ratio from 16-20 cells using MitoQC counter (FIJI), comparing VEH to DFP. P-values from Student’s t-test (** p < 0.0001). (E) Western blotting results for BNIP3 and ACTIN after VEH or DFP treatment overnight in MC3T3E1C4 cells.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A) Calvarial osteoblasts (COB) from Nondiff (top) or 21 days in osteogenic media (DIFF, bottom panels). The red punctae indicate mitophagy. (B) Quantification of mitolysosomes/cell area from ≥30 cells using MitoQC counter (FIJI), comparing nondifferentiated cells to cells differentiated for 7, 14 or 21d, n=3 experiments. P-values from one-way ANOVA followed by Tukey’s post-hoc test (**** p < 0.0001). (C) RT-qPCR on preosteoblasts compared to cells that have been differentiated for different timepoints D0 compared to D7, D14 and D21 of osteogenic differentiation including, Bnip3 , Bnip3l , Bcl2l13 and Pink1 . P-values from one-way ANOVA followed by Tukey’s post-hoc test (*p < 0.05 **p < 0.01, ***p < 0.001 **** p < 0.0001). ( D) MC3T3E1C4 cells treated with VEH (left, top panel) and CoCl2 (200uM, Right panel). The different panels indicate DAPI (blue), FITC Green (BNIP3 staining), Mitotracker (Red) and overlay. Representative confocal images from n =3 experiments. (E) Quantification of Rod/branch lengths using MiRA plugin from ImageJ showing a significant decrease in networking after CoCl 2 treatment. n=5-6 cells, P-values from students t-test (** p < 0.0001). (F) OCR (Top panel) and ECAR (bottom panel) after CoCl 2 (red line); blue line is control (each with n>20 wells/group). There is a significant decrease in basal OCR with a compensatory increase in ECAR rates as indicated in the ECAR panel before oligomycin treatment. Asterisks indicate statistically significant P-values using Student’s t-test.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Quantitative RT-PCR, Staining, Control

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A) RT-qPCR of AdshRNAi Bnip3 KD cells compared to scrambled control (n=3 replicates) on day 14 of differentiation BNIP3 KD significantly reduces expression of osteogenic and stress-response genes, including Bnip3 , Bnip3l , Bcl2l13 , Atf4 , Mfn2 , Drp1 , Alp l, Col1a1 , Runx2 , Sp7 and Ocn , normalized to Hprt . Asterisks indicate p- values from student’s t-test were significantly different. (B) Mineralization assay using an OsteoImage Mineralization Assay Lonza fluorescence assay kit comparing Scr control vs AdshRNAi Bnip3 KD showing representative brightfield (left) and fluorescence (right) images. (C) Quantification of mineralization assay shown in B. Asterisks indicate P-values from Student’s t test (***p < 0.001). (D) PCA of proteomic data from AdshRNAi Bnip3 KD and Scr controls cells. BigOmics Analytical software was utilized to perform principal component analysis resulting in separate clusters demonstrating a distinct proteomic shift driven by loss of BNIP3. Replicates cluster separately within their respective groups. (E) Volcano plot of differentially expressed proteins analyzed by mass spectrometry reveals significant up and down-regulated proteins (highlighted in red and blue, respectively) following BNIP3 KD. (F) Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes reveals significant alterations in pathways related to protein localization to organelles, mitochondrial inner membrane organization, intracellular protein transport, translational initiation, regulation of translation, and cellular metabolic processes. Dot size reflects gene count and color indicates false discovery rate (FDR).

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Quantitative RT-PCR, Control, Expressing, Mineralization Assay, Fluorescence, Software, Mass Spectrometry, Membrane

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A,B) Shown are representative μCT images of male femora showing trabecular and cortical bone. (C) Quantification of bone measurements comparing effects of sex (n=5-7 males, n=5 females) and genotype in 16 week old mice. Trabecular bone volume, trabecular bone mineral density, trabecular thickness, cortical area, total periosteal area, and polar moment of inertia were reduced in males lacking Bnip3 compared to their wildtype littermates. P-values from 2 way-ANOVA are shown. ns = non-significant.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques:

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A) In WT and Bnip3 -/- males, trabecular %bone volume/total volume, trabecular number and trabecular spacing, osteoblast number per bone surface and osteoclast numbers per bone surface were quantified in the femur. Graphed are means ± SEM from 16 week old male mice, n=5-7 males, P-values from student’s t-test are shown. ns, not significant. (B) Representative image of trichrome staining of femora from WT and Bnip3 -/- mice. Images shown are representative of at least n=3 each.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Staining

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A) OCR (Top) and (B) ECAR (bottom) after Bnip3 KD (red line); blue line is scrambled control (representative of 3 trials, each with n>20 wells/group). There is increased proton leak and decreased FCCP induced OCR. There is a significant increase in basal ECAR rates as indicated in the ECAR panel before oligomycin treatment. Asterisks indicate statistically significant P-values using Student’s t-test.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Control

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: ( A) Mitophagy in Wildtype (WT) and Bnip3 -/- (KO) bone marrow stromal cells on a MitoQC background isolated from male mice at 6-8 wk old weeks of age. Images shown are representative of two different isolations using at least 3 animals each. BMSCs from WT and Bnip3 -/- MitoQC mice treated with non-differentiation (left panels) and 7days osteogenic differentiation (right panel). The red punctae indicate mitophagy in the top panels WT non differentiation and WT 7days and Bnip3 -/- non differentiation and Bnip3 -/- 7 days osteogenic differentiation (bottom panels). Representative confocal images from n=3 samples. (B) Quantification of Red/Green ratio from ≥45 cells each using MitoQC counter (FIJI), comparing WT non differentiation and WT 7days and Bnip3 -/- non differentiation and Bnip3 -/- 7 days osteogenic differentiation. P-values from one-way ANOVA followed by Tukey’s post-hoc test (** p < 0.0001). (C) Western blotting analysis identified an increase in p53 protein expression in Bnip3 KD cells. ACTIN was used as the loading control.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Isolation, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Impaired mitochondrial stress signaling mediates bone loss in male mice in the absence of BNIP3

doi: 10.64898/2026.04.06.710936

Figure Lengend Snippet: (A) Principal component analysis (PCA) of global gene expression profiles from Bnip3 KD ( Bnip3 , n=4) and scrambled control (Scr, n=4) MC3T3E1C4 samples. PC1 accounts for 98.75% of the total variance and clearly separates BNIP3-deficient cells from controls, indicating a strong and consistent genotype-dependent transcriptional signature. (B) Protein–protein interaction network of significantly altered genes generated using STRING analysis. Several key regulatory nodes, including Atf5 , Gadd45Aa , Asns , Akt2 , Pycr1 , and Slc6a9 , form a highly interconnected network associated with mitochondrial stress responses, amino acid metabolism, cell-cycle regulation, and survival pathways. (C) Gene Ontology enrichment (Biological Process) of BNIP3 regulated genes. Top enriched categories include regulation of metabolic processes including α-amino acid metabolic processes, α-amino acid biosynthetic process, Carboxylic acid metabolic and catabolic processes. Circle size represents gene count and color indicates false discovery rate (FDR). (D) KEGG pathway enrichment analysis of differentially expressed genes. Significantly enriched pathways include Wnt signaling, HIF-1 signaling, p53 signaling, MAPK signaling, ECM receptor interaction, focal adhesion, cell cycle, DNA replication, and cancer-associated pathways. The size of each bubble represents the number of genes involved and color intensity corresponds to statistical significance (q-value). (E) BNIP3 KD blunts ATF4 and ATF5 activation. Western blot analysis of MC3T3E1C4 cells with Bnip3 KD compared to Scr controls showed a significant decrease in ATF4 and ATF5 levels. ACTIN was used as a loading control. (F) Oxygen consumption rates measured by XF96 analyzer from cells treated with DMSO, tunicamycin (2μM), bafilomycin (25nM) and FCCP (0.5µM). Asterisks indicate P-values from student’s t-test. (G) Oxygen consumption rates measured by XF96 analyzer show a decrease when cells were treated with rotenone (Complex I) (12μM), antimycin (Complex III) (7.6μM) and oligomycin (Complex V) (1µM). Asterisks indicate P-values from student’s t-test were significantly different between groups. (H) Scrambled and Bnip3 KD cells treated overnight with rotenone (12μM) and antimycin (7.6μM), result in a stress response with an increase in ATF4 in control cells but not when BNIP3 levels are reduced, suggesting that stress responses in response to mitochondrial dysfunction require BNIP3 to transduce this signal to ATF4. Scrambled and Bnip3 KD cells treated overnight with bafilomycin, tunicamycin, FCCP and Oligomycin which can trigger ER and lysosomal specific and mitochondrial stress response with an increase in ATF4. This response is relatively normal in the Bnip3 KD cells with some effects from Bafilomycin and Tunicamycin suggesting that the dysfunction could partially be related to other organellar stress response. Actin is shown as loading control for each condition.

Article Snippet: Adenovirus mediated short hairpin RNA (shRNAi) against mouse Bnip3 (Ad-sh- Bnip3 ) and a negative control scrambled shRNAi (Ad-sh-Scr) were purchased from Vector Biolabs (PA, USA).

Techniques: Gene Expression, Control, Generated, Activation Assay, Western Blot, Transduction

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Scatter plots of genes ranked by Pearson’s correlation with Ctsl expression across lung tumor and skeletal muscle (TB versus CON). B Comparative ranking of these correlation coefficients in lung tumor versus muscle for Ctsl ’s direct neighbors in PPI network (as shown in Fig. ). C Correlation of Ctsl versus Bnip3 expression in lung tumor and muscle (log 2 TPM + 1). D Three-way Venn diagram of Ctsl ’s direct neighbors that are upregulated in lung tumor, upregulated in muscle (TB versus CON), and downregulated in muscle (αCD8a versus IgG). Subnetwork of Ctsl - Bnip3 neighbors upregulated in tumors ( E , TB versus CON; node color represents log 2 FC). Bar plot ( F ) of KEGG pathways enriched among genes (adjusted p -value < 0.05). Subnetwork of Ctsl - Bnip3 neighbors ( G ) upregulated in muscle (left in the box: TB versus CON) and downregulated in muscle (right in the box: αCD8a versus IgG group). Node color indicates log 2 FC. Bar plots ( H ) of KEGG pathways enriched among genes (adjusted p -value < 0.05). For C , two-sided t -test for Pearson correlation; for F and H , hypergeometric test with Benjamini–Hochberg correction. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were transfected with 20 nM of siRNAs against Ctsl (SR411229, OriGene Technologies, Rockville, MD, USA) or Bnip3 (SR404627, OriGene Technologies) using Lipofectamine RNAiMax (13778150, Invitrogen) for 24 h. Then, C2C12 myotubes were co-cultured with splenic CD8 + T cells in the presence of either DM or CM for an additional 72 h.

Techniques: Expressing

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Human PPI network of CTSL-BNIP3 neighbors colored by log 2 FC (tumor versus matched normal lung) in TCGA LUAD. B Immunofluorescence of CTSL (green) and BNIP3 (red) in LUAD tissue microarray cores: normal adjacent, n = 22; tumor cores, n = 128. Bar graph shows proportions of BNIP3-high/-low within the CTSL-high and CTSL-low tumors. C Kaplan–Meier overall survival for TCGA LUAD stratified by combined CTSL and BNIP3 expression ( CTSL-BNIP3 -high, red, n = 183; CTSL-BNIP3 -low, black, n = 156). D GSEA plots of ALONSO_METASTASIS_UP in CTSL-BNIP3 high versus low groups in TCGA LUAD (left) and in TB mice (right). E Representative WB of indicated proteins in TB muscle. Two mice per group are shown ( n = 4 per group); quantification of all four mice in Supplementary Fig. . Samples derive from the same experiment; different gels for Ctsl, N-Cad, Gapdh, and another for Bnip3 were processed in parallel. F WB of indicated proteins in A549 cells treated with or without CTSLi; GAPDH as loading control; quantification from four independent biological replicates in Supplementary Fig. . Samples derive from the same experiment; different gels for CTSL and another for N-Cad, Bnip3, GAPDH, were processed in parallel. Migration ( G ) and invasion ( H ) assays for A549 cells treated with or without CTSLi. Bar graphs represent quantified gap closure ( G ) and invaded cells ( H ); biological triplicates per group. WB of indicated proteins in A549 cells transfected with si-CTSL ( I ) or si-BNIP3 ( J ); quantification from four independent biological replicates in Supplementary Fig. . For I , samples derive from the same experiment; different gels for CTSL and another for N-Cad, Bnip3, GAPDH, were processed in parallel. For J , samples derive from the same experiment; different gels for CTSL, N-cad, GAPDH, and another for BNIP3 were processed in parallel. Quantified migration after si-CTSL ( K ) or si-BNIP3 ( L ) transfection; biological triplicates per group. M Working model: combined αPD-L1 and CTSLi therapy suppresses tumorigenesis. For B , one-sided Fisher’s exact test; for C , log-rank test; for D , GSEA; for G , H , K , L , data are mean ± SEM, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were transfected with 20 nM of siRNAs against Ctsl (SR411229, OriGene Technologies, Rockville, MD, USA) or Bnip3 (SR404627, OriGene Technologies) using Lipofectamine RNAiMax (13778150, Invitrogen) for 24 h. Then, C2C12 myotubes were co-cultured with splenic CD8 + T cells in the presence of either DM or CM for an additional 72 h.

Techniques: Immunofluorescence, Microarray, Expressing, Control, Migration, Transfection

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Human PPI network of CTSL-BNIP3 neighbors colored by log 2 FC in skeletal muscle from CTSL-BNIP3 -high versus -low individuals (GTEx). B GSEA of DEGs in GTEx muscle: top five positively and negatively enriched pathways (NES, normalized enrichment scores). C Representative GSEA plots for WP_PROTEASOME_DEGRADATION (top) and WP_ELECTRON_TRANSPORT_CHAIN_OXPHOS_SYSTEM_IN_MITOCHONDRIA (bottom) in CTSL-BNIP3 -high versus -low muscles. D Representative WB of indicated proteins in TB muscle. Two mice per group are shown ( n = 4 per group); quantification of all four mice in Supplementary Fig. . Samples derive from the same experiment; different gels for Ctsl, another for Bnip3, another for vinculin, were processed in parallel. E WB and quantification of Bnip3 in C2C12 myotubes 24 h after si-Ctsl or si-Cont transfection; biological triplicates for each group. Vinculin as a loading control. F C2C12 myotube diameter after 24 h transfection with si-Bnip3 or si-Cont and 72 h co-culture with CD8 + T cells in CM; 12 myotubes per well, biological triplicates for each group. Relative mtDNA copy number in GA muscle from TB mice ( G , n = 8); in C2C12 myotubes co-cultured with CD8 + T cells in CM plus or minus CTSLi ( H , biological triplicates for each group); and transfected with si-Ctsl, si-Bnip3, or si-Cont then co-cultured with CD8 + T cells in CM ( I , biological triplicates for each group). C2C12 myotube diameter ( J ) and mtDNA copy number ( K ) after co-culture with CD8 + T cells in CM plus or minus mouse recombinant Gzma (10 ng/ml); biological triplicates for each group. L Working model illustrating how the CTSL-BNIP3 network promotes muscle wasting in cooperation with cytotoxic T cells. For B and C , GSEA for functional enrichment analysis; for E – K , data are mean ± SEM, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were transfected with 20 nM of siRNAs against Ctsl (SR411229, OriGene Technologies, Rockville, MD, USA) or Bnip3 (SR404627, OriGene Technologies) using Lipofectamine RNAiMax (13778150, Invitrogen) for 24 h. Then, C2C12 myotubes were co-cultured with splenic CD8 + T cells in the presence of either DM or CM for an additional 72 h.

Techniques: Muscles, Transfection, Control, Co-Culture Assay, Cell Culture, Recombinant, Functional Assay

Journal: Biomolecules

Article Title: Modulation of Carnitine Palmitoyl Transferase 1b Expression and Activity in Muscle Pathophysiology in Osteoarthritis and Osteoporosis

doi: 10.3390/biom14101289

Figure Lengend Snippet: Bnip3 localization in cultured myoblasts from CTR, OA, and OP patients. Immunofluorescence for Bnip3 in cultured myoblasts from CTR, OA, and OP patients (scale bar = 100 µm). Specifically, in CTR myoblasts, the protein is diffuse along all the cytoplasm, whereas in OA myoblasts, Bnip3 is aggregated in numerous and distinct spots (puncta) similar to autophagosomes (white arrowheads). OP myoblasts revealed a lower expression of Bnip3 with no evident puncta.

Article Snippet: For immunohistochemical analysis, 3 μm-thick muscle sections were incubated with polyclonal rabbit anti-Cpt1b antibody (Thermo Fisher Scientific, Waltham, MA, USA), monoclonal mouse anti-BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) antibody (Thermo Fisher Scientific, Waltham, MA, USA), and monoclonal mouse anti-Fast Myosin Skeletal Heavy chain antibody [MY-32] (ab51263, Abcam, Cambridge, UK).

Techniques: Cell Culture, Immunofluorescence, Expressing

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) BNIP3 and NIX are stabilised in PPTC7-deficient U2OS cells. CRISPR-CAS9 was used to knockout PPTC7 in U2OS cells. Cells were treated with cycloheximide for the indicated times before immunoblotting as indicated. The red asterisks indicate PPTC7-specific bands at 28 kDa, 32 kDa and 40 kDa. ns = non-specific band at ~36 kDa. ( B ) Immunofluorescence staining of BNIP3 and NIX demonstrating increased levels of both proteins at mitochondria in PPTC7-deficient cells U2OS cells. Cells were stained with BNIP3 or NIX (in green) and counterstained with MitoTracker (in magenta). ( C ) Re-expression of PPTC7 into PPTC7-deficient cells reduces the stability of BNIP3 and NIX. PPTC7-HA was transduced into PPTC7-deficient 293T cells. The half-lives of BNIP3 and NIX were analysed by immunoblotting after a cycloheximide chase. ( D ) Targeting PPTC7 and FBXL4 simultaneously does not further increase BNIP3 and NIX levels or stability. PPTC7 was sequentially knocked out of previously generated FBXL4-deficient cells to make the double knockout of PPTC7 and FBXL4 (dKO FBXL4/PPTC7). The 32 kDa band of PPTC7 was upregulated in the FBXL4 KO cells. ( E ) FBXL4 requires PPTC7 to mediate the downregulation of NIX. FBXL4-HA was transduced in parental, FBXL4 KO, PPTC7 KO, and FBXL4/PPTC7 dKO cells. NIX protein levels (magenta) were monitored in FBXL4(HA)-expressing cells (green). Full figure is provided in Appendix Fig. S . Scale bar = 20 microns. ( F ) PPTC7 requires FBXL4 to mediate the downregulation of NIX. PPTC7-HA was expressed in parental, FBXL4 KO, PPTC7 KO, and FBXL4/PPTC7 dKO cells. NIX protein levels (magenta) were monitored in PPTC7(HA) expressing cells (green). To correlate NIX levels with PPTC7 expression, PPTC7 expressing cells (green) are outlined in orange dotted lines. Full figure is provided in Appendix Fig. S . Scale bar = 20 microns. ( G ) FBXL4 is required for the PPTC7-mediated downregulation of BNIP3 and NIX. PPTC7(HA) was transduced into U2OS, FBXL4 KO or PPTC7 KO cells and the half-lives of BNIP3 and NIX were monitored by immunoblotting. PPTC7(HA) rescue into PPTC7 KO cells causes a reduction of BNIP3 and NIX. In contrast, the expression of PPTC7(HA) into FBXL4 KO cells does not cause a reduction in either NIX or BNIP3. ( H ) PPTC7 deficiency leads to increased mitophagy in basal conditions and after DFP treatment. U2OS mt-Keima-PPTC7 KO cells were treated with DFP for 24 h. Emission signals at neutral pH were obtained after excitation with the 458 nm laser (green), and emission signals at acidic pH were obtained after excitation with the 458 nm laser 561 nm laser (magenta). Mitophagy is represented as the ratio of mt‐Keima 561 nm fluorescence intensity to mt‐Keima 458 nm fluorescence intensity for individual cells normalised to the mean of the untreated condition. Translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicates +/− standard deviation. P values were calculated based on the mean values using a one-way ANOVA. n = 3. Data Information: ( A , C , D ) The red asterisks indicate PPTC7-specific bands at 28 kDa, 32 kDa and 40 kDa. ns = non-specific band at ~36 kDa. ( G ) The orange asterisks denote PPTC7(HA) expression. ( B , E – H ) Scale bar of main = 20 microns. ( B ) Scale bar of inset (yellow boxes) = 1 micron. .

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: CRISPR, Knock-Out, Western Blot, Immunofluorescence, Staining, Expressing, Generated, Double Knockout, Fluorescence, Standard Deviation

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) Distinct guide RNAs targeting PPTC7 result in BNIP3 and NIX upregulation. Cells were transfected PPTC7 guide 1 (G1) or guide 2 (G2). ( B ) BNIP3 and NIX are stabilised in PPTC7-deficient 293T cells. Cells were treated with cycloheximide for the indicated times before immunoblotting as indicated. ( C ) Inhibition of HIF1α with echinomycin does not prevent the accumulation of BNIP3 and NIX in PPTC7-deficient cells. U2OS cells or PPTC7 KO cells were treated with echinomycin for 24 h. Echinomycin completely prevented the DFP-induced upregulation of BNIP3 and NIX, but only partially prevented the accumulation of BNIP3 and NIX in PPTC7 KO cells. DFP induces the 32 kDa form of PPTC7. ( D ) FBXL4 requires PPTC7 for its ability to promote BNIP3 and NIX turnover. FBXL4-HA was expressed in parental, FBXL4 KO, PPTC7 KO, and FBXL4/PPTC7 dKO cells. BNIP3 and NIX protein levels were monitored by Western blotting in response to FBXL4 expression. ( E ) PPTC7-mediated downregulation of BNIP3 and NIX does not occur in FBXL4-deficient cells. PPTC7-FLAG was transfected into either PPTC7 KO or FBXL4 KO cells. Cells were fixed and stained for FLAG(PPTC7) (green) and either NIX or BNIP3 (magenta). The orange dotted line surrounds the cells that have been transfected with PPTC7. ( F ) PPTC7(HA) overexpression causes the downregulation of BNIP3 and NIX in U2OS and 293T cells in basal conditions and after DFP treatment. PPTC7(HA) was transduced into U2OS or 293T cells and the levels of BNIP3 and NIX were monitored by immunoblotting. ( G ) PPTC7 overexpression results in the downregulation of BNIP3 and NIX in basal conditions as well as after DFP treatment. U2OS cells or U2OS cells stably transfected with PPTC7(HA) were treated with DFP for 24 h. Cells were subjected to cycloheximide chase. s.e. = shorter exposure. ( H ) PPTC7 overexpression suppresses DFP-induced mitophagy. Mitophagy was assessed U2OS mt-Keima cells or U2OS mt-Keima cells overexpressing PPTC7(HA) in the presence or absence of DFP. Emission signals at neutral pH were obtained after excitation with the 458 nm laser (green), and emission signals at acidic pH were obtained after excitation with the 458 nm laser 561 nm laser (magenta). Mitophagy is represented as the ratio of mt-Keima 561 nm fluorescence intensity divided by mt-Keima 458 nm fluorescence intensity for individual cells normalised to the mean of the untreated U2OS cells. Translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicates +/− standard deviation. P values were calculated based on the mean values using a one-way ANOVA. **** P <0.0001. n = 3 independent experiments. Data Information: ( E , H ) Scale bar = 20 microns. ( B , C ) The red asterisks indicate PPTC7-specific bands and 28 kDa, 32 kDa, and 40 kDa. Arrow and ns=non-specific band at ~36 kDa.

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Transfection, Western Blot, Inhibition, Expressing, Staining, Over Expression, Stable Transfection, Fluorescence, Standard Deviation

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) The 32 kDa migrating form of PPTC7 is located on the mitochondrial outer membrane, while the lower migrating form of PPTC7 resides in the matrix. Mitochondria were isolated from parental U2OS, FBXL4 KO, PPTC7 KO or PPTC7/FBXL4 dKO cells. The submitochondrial localisation of PPTC7 was determined by proteinase K treatment, which degrades proteins that are not imported within the mitochondria, i.e., outer membrane proteins. ( B ) The outer membrane form of PPTC7 is induced by DFP treatment and in FBXL4-deficient cells. U2OS cells or FBXL4 KO cells were treated with DFP for the indicated times. ( C ) Analysis of the stability of the outer membrane form of PPTC7 in response to FBXL4 deficiency. FBXL4 KO cells and FBXL4 KO cells expressing FBXL4(HA) were subjected to cycloheximide chase assay in the presence or absence of DFP. DFP treatment and FBXL4 deficiency both extended the half-live of OM-PPTC7. ( D ) PPTC7 interacts with NIX/BNIP3 in basal conditions in an FBXL4-independent manner. U2OS cells or FBXL4 KO cells were transfected with PPTC7-FLAG and treated with DFP for 24 h. Cell lysates were immunoprecipitated with anti-FLAG beads, and the immuno-precipitates were analysed by immunoblotting as indicated. WCL whole-cell lysates. ( E ) PPTC7 interacts with FBXL4. As in ( E ), including PPTC7-FLAG immunoprecipitations in FBXL4 KO cells rescued with HA-tagged FBXL4 WT. ( F ) AlphaFold2 multimer model of FBXL4, PPTC7 and NIX. See also Fig. S . ( G ) The FBXL4 pocket is coloured to indicate surface sequence conservation or surface electrostatics using the Consurf or Protein-Sol Patches server. Data Information: ( A – C ) The red asterisks indicate PPTC7-specific bands detected by immunoblotting. The arrow points to the non-specific band (ns) at 36 kDa which is enriched in mitochondria. ( D , E ) The orange asterisks denote PPTC7(FLAG) expression. .

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Membrane, Isolation, Expressing, Transfection, Immunoprecipitation, Western Blot, Sequencing

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) Analysis of the stability of the outer membrane form of PPTC7 in response to DFP treatment, DMOG treatment, and/or FBXL4 deficiency. ( B ) Analysis of the stability of the outer membrane form of endogenous PPTC7 and exogenous PPTC7(HA) in response to DFP treatment. ( C ) PPTC7 is not required for FBXL4 to interact with CUL1 or SKP1. 293T or 293T PPTC7 KO cells were transfected with either FLAG-FBXL4 or FLAG-PPTC7. Cells were treated with MLN4924 for 24 h where indicated. Cell lysates were immunoprecipitated with anti-FLAG beads, and the immuno-precipitates were analysed by immunoblotting as indicated. FBXL4 binds to CUL1 and SKP1, whereas PPTC7 binds to BNIP3 and NIX. WCL = whole-cell lysates. Red asterisks mark the PPTC7 transfected product and magenta asterisks mark the FBXL4 transfected product. ( D ) ITC comparison of PPTC7 wild-type binding to Mg 2+ and Mn 2+ . The binding affinities were 6.21 nM for Mn 2+ and 237 nM for Mg 2+ . ( E ) ITC comparison of PPTC7 wild-type binding to BNIP3 and NIX in the presence of Mn 2+ . The binding affinities were 20.1 μM for BNIP3 and 37.5 μM for NIX. ( F ) Overlay of the top 5 AlphaFold2 models of FBXL4, PPTC7, NIX with predicted aligned error (PAE) plots. ( G ) Overlay of the top 5 AlphaFold2 models of FBXL4, PPTC7, BNIP3 with PAE plots.

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Membrane, Transfection, Immunoprecipitation, Western Blot, Comparison, Binding Assay

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) AlphaFold2 model of PPTC7 with active site residues indicated. This is based on a comparison to the PBCP structure (PDB ID 6AE9). The three putative metal ions and the incoming phosphate group are modelled based on an alignment of the PPTC7 and PBCP structures. ( B ) Binding to BNIP3 and NIX is preserved in the PPTC7-D290N mutants. PPTC7 KO 293T cells expressing FBXL4(HA) were transfected with PPTC7-FLAG or D78N, D223N, and D290N mutants. Cells were treated with DFP for 24 h where indicated. Cell lysates were immunoprecipitated with anti-FLAG beads, and the immuno-precipitates were analysed by immunoblotting as shown. ( C ) PPTC7-D290N can restore the turnover of BNIP3 and NIX to a similar extent as wild-type PPTC7. The PPTC7 KO cells were rescued with wild-type PPTC7-FLAG or PPTC7-D78N, PTPC7-D223N or PPTC7-D290N variants. Cells were treated with cycloheximide for 3 h before harvesting. Samples were lyzed, and immunoblotting was performed. s.e. short exposure. ( D ) PPTC7-D290N can rescue the degradation of BNIP3 and NIX to a similar extent as wild-type PPTC7. NIX and BNIP3 protein levels (magenta) were monitored in PPTC7(FLAG) expressing cells. To correlate BNIP3/NIX levels with PPTC7 expression, PPTC7 expressing cells (green) are outlined in orange dotted lines. Scale bar = 20 microns. ( E ) PPTC7 wild-type can dephosphorylate BNIP3 but PPTC7-D290N cannot. Electrophoretic mobility shift assays of BNIP3 using phos-tag gels were performed to assess the phosphorylation status of BNIP3. Purified PPTC7 or PPTC7-D290N were incubated with 293T PPTC7 KO cell lysates. Wild-type PPTC7 could dephosphorylate BNIP3 in a Mn2+-dependent manner as demonstrated by the loss of the upper form of BNIP3 (arrow). However, PPTC7-D290N was not able to dephosphorylate BNIP3 in the same conditions. Lamda-phosphatase is used as a control to demonstrate that the upper band of BNIP3 seen on the phos-tag gels is the phosphorylated species. ( F ) In vitro pNPP dephosphorylation assay using purified PPTC7 wild-type and PPTC7-D290N. Top Panel: The dephosphorylation of pNPP was measured at 405 nm after a 15-min reaction using increasing concentrations of PPTC7. Bottom panel: 10 mM pNPP was added to 4 µM PPTC7 in the presence or absence of either MnCl 2 or MgCl 2 . Bars represent mean values +/− standard deviation. n = 1, individual data points represent technical replicates. Data Information: ( B , C ) The orange asterisks denote PPTC7(FLAG) expression. .

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Comparison, Binding Assay, Expressing, Transfection, Immunoprecipitation, Western Blot, Electrophoretic Mobility Shift Assay, Phospho-proteomics, Purification, Incubation, Control, In Vitro, De-Phosphorylation Assay, Standard Deviation

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) Sequence alignment of PPTC7 orthologues with active site residues from Fig. indicated. ( B ) Disruption of the PPTC7’s active site residues from aspartate to alanine compromises its ability to downregulate BNIP3 and NIX. PPTC7 KO cells were transduced with PPTC7 wild-type, PPTC7-D78A, or PPTC7-D223A/D78A. Wild-type PPTC7 rescued the turnover of BNIP3 and NIX, however, the D78A and D223A/D78A variants did not. ( C ) Aspartate to alanine mutations in PPTC7’s active cannot rescue the downregulation of NIX by PPTC7. PPTC7 KO cells were complemented with PPTC7 or active site mutants and NIX levels were analysed by immunofluorescence microscopy. Scale bar = 20 microns. ( D ) Disruption of the PPTC7’s active site residues from aspartate to alanine interferes with its ability to bind to BNIP3 and NIX. Cell lysates expressing PPTC7(FLAG) and mutants were immunoprecipitated with anti-FLAG beads, and the immuno-precipitates were analysed by immunoblotting as shown. Unlike the PPTC7-D78N mutant in Fig. , the D78A mutant is unable to bind to BNIP3 or NIX.

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Sequencing, Disruption, Transduction, Immunofluorescence, Microscopy, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) AlphaFold2 prediction of the PPTC7–NIX complex. Key residues in the PPTC7 interface are highlighted in cyan. ( B ) PPTC7-Y179D and PPTC7-N181E variants are unable to interact with BNIP3 and NIX. PPTC7(FLAG) and specified mutants were transfected into U2OS cells. Cells were treated with DFP for 24 h. Cell lysates were precipitated with anti-FLAG affinity resin and the immuno-precipitates were analysed by immunoblotting. ( C ) PPTC7-Y179D and PPTC7-N181E variants are unable to mediate the turnover of BNIP3 and NIX. PPTC7(HA)-WT, PPTC7-Y179D or PPTC7-N181E were expressed in PPTC7 KO 293T cells. BNIP3 and NIX stability was assessed using a cycloheximide chase. ( D ) AlphaFold2 prediction of the PPTC7–NIX complex. Key residues in the NIX interface including Arg147 are highlighted. ( E ) Residues W144 and RPE 147-149 in NIX are critical for binding to OM-PPTC7. PPTC7(HA) was transduced into HeLa Flp-in BNIP3/NIX double KO cells stably expressing inducible FLAG-tagged NIX-WT or NIX mutants. NIX expression was induced with doxycycline for 24 h. Cell lysates were immunoprecipitated with anti-FLAG beads, and the immuno-precipitates were analysed by immunoblotting. The levels of NIX-W144D and NIX-RPE/AAA were significantly higher than NIX-wild-type. Red asterisks mark the NIX(FLAG) proteins. The different migration of the point mutants by electrophoresis could be due to the change in charge of the residues or because PPTC7 is unable to dephosphorylate NIX. ( F ) NIX-W144D and NIX-RPE/AAA are expressed at higher levels in comparison to wild-type NIX. HeLa Flp-In BNIP3/NIX double knockout Keima cells stably expressing NIX and NIX mutants were treated with doxycycline for 48 h. Cells were subjected to a cycloheximide chase. ( G ) Expression of hyper-stable NIX-W144D and NIX-RPE/AAA leads to an increase in steady-state mitophagy compared with NIX-wild-type. HeLa Flp-In BNIP3/NIX double knockout Keima cells stably expressing NIX and NIX mutants were treated with doxycycline for 48 h and mitophagy was evaluated using live-cell confocal fluorescence microscopy. Mitophagy is represented as the ratio of mt-Keima 561 nm fluorescence intensity divided by mt-Keima 458 nm fluorescence intensity for individual cells normalised to mitophagy observed in cells expressing NIX-wild-type. Translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicates +/− standard deviation. P values were calculated based on the mean values using a one-way ANOVA. .

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Transfection, Western Blot, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Migration, Electrophoresis, Comparison, Double Knockout, Fluorescence, Microscopy, Standard Deviation

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) Sequence alignment of BNIP3 and NIX orthologues. Functionally relevant motifs or domains are indicated. BNIP3 accession Q12983 (194 aa) replaces the previous BNIP3 accession EAW49143.1 (259 aa) characterised previously (Nguyen-Dien et al, ). ( B ) ITC comparison of PPTC7-D290N and PPTC7-D290N/Y179D binding to NIX peptide. The binding affinities were 35.9 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± 1.09 for PPTC7-D290N and non-binding for PPTC7-D290N/Y179D. ( C ) PPTC7-Y179D and PPTC7-N181E variants localise to mitochondria. Wild-type PPTC7 can reduce NIX levels when expressed in PPTC7 KO cells, but PPTC7-Y179D and PPTC7-N181E cannot. ( D ) Arg147 in NIX is critical for binding to PPTC7. PPTC7(HA) was transduced into cell lines expressing inducible NIX mutants. NIX expression was induced with doxycycline for 24 h. Cell lysates were immunoprecipitated with anti-FLAG beads, and the immuno-precipitates were analysed by immunoblotting. ( E ) Arg147 in NIX is critical for its turnover. HeLa Flp-in BNIP3/NIX double KO cells expressing FLAG-tagged NIX-WT or NIX binding mutants (FLAG-tagged NIX Δ144-150, NIX-RPE-AAA and NIX-RPE-DAA) were subjected to a cycloheximide chase. ( F ) Expression of NIX-RPE/AAA and NIX-RPE/DAA in NIX leads to an increase in basal levels of mitophagy compared with NIX-wildtype. Hela Flp-In NIX knockout/ Hela Flp-In BNIP3/NIX double knockout Keima cells stably expressing NIX mutants were treated with doxycycline for 48 h and mitophagy was evaluated using live-cell confocal fluorescence microscopy. Translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicates +/− standard deviation. P values were calculated based on the mean values using a one-way ANOVA. Data Information: ( C , F ) Scale bars = 20 microns.

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Sequencing, Comparison, Binding Assay, Expressing, Immunoprecipitation, Western Blot, Knock-Out, Double Knockout, Stable Transfection, Fluorescence, Microscopy, Standard Deviation

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) Alphafold2 structural modelling of FBXL4 in complex with PPTC7. Alphafold2 predicts a high-confidence interaction between FBXL4 and PPTC7 centred on Met71 and Arg544 in FBXL4. ( B ) Met71 and Arg544 in FBXL4 are involved in the interaction with PPTC7. FBXL4 knockout cells were complemented with HA-tagged wild-type FBXL4, FBXL4-M71E, FBXL4-R544E or FBXL4-M71E/R544E. Cells were transfected with FLAG-tagged PPTC7 as indicated, lysed, and subjected to affinity purification using anti-FLAG resin. WCL whole-cell lysates. ( C ) FBXL4-M71E, FBXL4-R544E or FBXL4-M71E/R544E variants are unable to mediate BNIP3 and NIX downregulation and destabilisation. U2OS FBXL4 KO cells were rescued with wild-type FBXL4(HA) or specified variants. ( D ) Localisation of FBXL4-M71E, FBXL4-R544E or FBXL4-M71E/R544E variants. U2OS FBXL4 KO cells expressing FBXL4(HA) wild-type or specified variants were fixed and stained for HA to detect FBXL4 (in magenta) or NIX (green). NIX levels are inversely correlated with the ability of FBXL4 to bind to PPTC7. Scale bar = 20 microns. ( E ) FBXL4-M71E/R544E is less efficient than wild-type FBXL4 in mediating mitophagy suppression. U2OS mt-Keima cells, U2OS mt-Keima FBXL4 KO cells and FBXL4 KO cells rescued with FBXL4 constructs were analysed by confocal microscopy to quantify mitophagy. P values were calculated based on the mean values using a one-way ANOVA. 4 individual data points outside axis limits. n = 3 independent replicates. Data Information: ( C ) Red asterisks mark the PPTC7-specific bands detected by immunoblotting. .

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Knock-Out, Transfection, Affinity Purification, Expressing, Staining, Construct, Confocal Microscopy, Western Blot

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: ( A ) Working model for the role of PPTC7 in mitophagy suppression. In steady-state conditions, low levels of PPTC7 localise at the mitochondrial outer membrane to mediate the constitutive turnover of BNIP3/NIX, and the majority of PPTC7 localises in the mitochondrial matrix. PPTC7 accumulates at the mitochondria outer membrane in conditions of high mitophagy such as pseudohypoxia to dampen mitophagy. PPTC7 accumulation on the outer membrane may be a result of active retention mechanisms or defective mitochondrial import. ( B ) Model of the combination of Alphafold2 PPTC7-BNIP3-FBXL4 with the structure of Skp1-Cul1-Ube2d-Ub-Nedd8 (Baek, , PDB ID 6TTU). PPTC7 interacts with BNIP3/NIX and with FBXL4. One interpretation of our data is that PPTC7 bridges the interaction between FBXL4 and BNIP3/NIX to position BNIP3/NIX substrates for productive ubiquitylation by SCF FBXL4 .

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Membrane

Journal: EMBO Reports

Article Title: PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCF FBXL4

doi: 10.1038/s44319-024-00181-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: BNIP3 (ANa-40) mouse monoclonal , Santa Cruz Biotechnology , sc-56167.

Techniques: Recombinant, CRISPR, Plasmid Preparation, Sequencing, Software