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