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: Molecular Metabolism

Article Title: Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

doi: 10.1016/j.molmet.2023.101717

Figure Lengend Snippet: Direct regulation of Bnip3 transcription by AhR. (A) The mRNA level of Bnip3 and Ahr in the GEO dataset (GSE46495) during fed and fasted conditions. (n = 5, each). (B) Expression levels of Bnip3, Ahr, and Cyp1a1 in fed and fasted livers of WT or AhR KO mice (n = 3 or 4). Data were shown as box and whisker plots. Box, interquartile range (IQR); whiskers, min to the max; and horizontal line within box, median, ∗p < 0.05, ∗∗p < 0.01. (C) Pearson's correlation between Ahr and Bnip3 mRNA levels. (D) BNIP3 protein levels in fed and fasted livers of WT and AhR KO mice (upper) and IHC for BNIP3 in fasted livers (lower, left). Representative images were shown (n = 3 each). Scale bar, 100 μm. The quantification of BNIP3 expression (lower, right) was measured by Image J. (E) Increased BNIP3 expression by endogenous AhR ligand, kynurenine (Kyn) treatment (100 μM, 24 h) in mouse primary hepatocyte (left) and AML12 cells (middle and right). (F) AhR recruitment to the Bnip3 genomic locus in TCDD-treated liver (GSE97634). (G) ChIP-PCR analysis of AhR binding to Bnip3 genomic locus. (H) Reporter assays using the pGL3-basic vector containing WT ARE or Mutated ARE. HEK293 cells transfected with mock or AhR overexpression vector together with the reporter vector and treated Kyn for 24h. Results was normalized to WT control. (D), (E), (G) and (H), Data represented the mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. (H), ## was compared to AhR overexpressed group.

Article Snippet: Antibodies against BNIP3 (nbp1-77683s), LC3A (NB100-2331), and LC3B (NB600-1384) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Whisker Assay, Binding Assay, Plasmid Preparation, Transfection, Over Expression, Control

Journal: Molecular Metabolism

Article Title: Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

doi: 10.1016/j.molmet.2023.101717

Figure Lengend Snippet: Restored mitophagy by BNIP3 overexpression. (A) Changes of LC3A protein levels by Bnip3 overexpression in AhR knockdown cells. AML12 cells were co-transfected with siCon, siAhR, or siAhR with Bnip3-overexpressing plasmids. Band intensities represent values relative to siCon. (B) Changes of mitochondria-associated LC3A levels. Cytosolic and mitochondrial fractions were subjected to Western blotting. Cox IV was used for the control of mitochondrial fraction. (C) and (D) Visualization of mitophagy using mt-Keima assay and quantification. (E) Dual staining of mitochondria (Mitotracker) and autophagy marker (LC3A). Representative images are presented. Scale bar, 50 μm. (F) Quantification of colocalization of LC3A with mitotracker. (G) Mitochondrial superoxide levels measured by MitoSOX™ in AML12 cells transfected with siCon, siAhR, or siAhR with Bnip3 overexpression plasmids. (H) Mitoplate S-1 assay to measure mitochondrial substrate utilization. AML12 cells were transfected with siCon, siAhR, or siAhR with Bnip3-overexpressing plasmids for 48 h. The change in metabolic rate for each substrate/intermediate of mitochondrial/glycolytic pathways was shown in a heatmap (left) and change of substrate utilization in TCA cycle were shown as a bar graph (right). (A),(B), (D–H), Data represented the mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. PPP; pentose phosphate pathway.

Article Snippet: Antibodies against BNIP3 (nbp1-77683s), LC3A (NB100-2331), and LC3B (NB600-1384) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Over Expression, Knockdown, Transfection, Western Blot, Control, Staining, Marker

Journal: Communications Biology

Article Title: TRIM2 inhibits apoptosis by ubiquitinating BNIP3 to protect the intestine against ischemia-reperfusion injury in mice

doi: 10.1038/s42003-025-08708-2

Figure Lengend Snippet: A , B The HA-tagged TRIM2 and Flag-tagged BNIP3 plasmids were co-transfected into HEK293T cells. The exogenous interaction between TRIM2 and BNIP3 was confirmed by co-immunoprecipitation and western blot analysis. C The endogenous interaction between TRIM2 and BNIP3 was detected by co-immunoprecipitation and western blot in Caco-2 cells subjected to CT or H/R treatment. D Representative confocal images of Caco-2 and IEC-6 cells demonstrated the co-localization and distribution of TRIM2 and BNIP3. E Flag-tagged BNIP3 plasmids were transfected into HEK293T cells, and lysates were collected from the HEK293T cells after 24 hours of transfection. The purified GST or GST-TRIM2 proteins were incubated with the collected lysates overnight. The direct interaction between TRIM2 and BNIP3 was verified by a GST-pulldown and western blot experiment, and the purity of GST-TRIM2 was assessed through Coomassie blue staining. F Full-length Flag-BNIP3 and a series of deletion mutants of GFP-TRIM2 were transfected into HEK293T cells. The interaction was verified by co-immunoprecipitation and western blot analysis. G A schematic representation of the plasmid generation for various deletion mutants of GFP-TRIM2 and their interaction with BNIP3. H Full-length GFP-TRIM2 and various deletion mutants of Flag-BNIP3 were co-transfected into HEK293T cells. The interaction between the two proteins was confirmed through co-immunoprecipitation and western blot analysis. I Various deletion mutants of Flag-tagged BNIP3 plasmids were transfected into HEK293T cells, and lysates were collected from the HEK293T cells after 24 hours of transfection. The purified GST or GST-TRIM2 proteins were incubated with the collected lysates overnight. The direct interaction between TRIM2 and the deletion mutants of Flag-BNIP3 was verified by GST-pull down and western blot experiment, and the purity of GST-TRIM2 was assessed through Coomassie blue staining. J Schematic representation of plasmid generation for various deletion mutants of Flag-BNIP3 and the interaction with TRIM2.

Article Snippet: The BNIP3 antibody (44060S) was purchased from Cell Signaling Technology.

Techniques: Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, Staining, Plasmid Preparation

Journal: Communications Biology

Article Title: TRIM2 inhibits apoptosis by ubiquitinating BNIP3 to protect the intestine against ischemia-reperfusion injury in mice

doi: 10.1038/s42003-025-08708-2

Figure Lengend Snippet: A Flag-BNIP3 and HA-Ub plasmids were co-transfected into HEK293T cells in the presence or absence of TRIM2. Polyubiquitination of BNIP3 was quantified by co-immunoprecipitation and western blot analysis. B The indicated plasmids were co-transfected into HEK293T cells for 24 hours. The absence and level of ubiquitination of BNIP3 were then detected by co-immunoprecipitation with anti-Flag tag antibodies and immunoblotting (IB) with the indicated antibodies. C Flag-BNIP3 and HA-Ub plasmids were co-transfected into HEK293T cells for 24 hours, with or without TRIM2. Polyubiquitination of BNIP3 was detected by IB with a specific antibody for Ub, K48 Ub, or K63 Ub after immunoprecipitation. D HEK293T cells were co-transfected with the indicated plasmids for 24 hours. The absence and level of ubiquitination of BNIP3 were then detected by co-immunoprecipitation with anti-Flag tag antibodies, and IB with the indicated antibodies. E The endogenous ubiquitination of BNIP3 in Caco-2 cells transfected with HA-TRIM2 or HA-Vector plasmids was detected by IB with the specific antibody for Ub, K48, or K63 after immunoprecipitation. F The endogenous BNIP3 ubiquitination in Caco-2 cells infected with Lenti-si-TRIM2 or Lenti-si-Control virus was detected by IB with the specific antibody for Ub, K48, or K63 after immunoprecipitation. G A graphical representation of the 12-point mutation site in BNIP3, in which all lysine residues (K) were replaced with arginine (R). H HEK293T cells were co-transfected with the indicated plasmids for 24 hours. The polyubiquitination sites of BNIP3 by TRIM2 were identified in the lysates of HEK293T cells by IB with an anti-HA antibody after immunoprecipitation with anti-Flag tag antibodies.

Article Snippet: The BNIP3 antibody (44060S) was purchased from Cell Signaling Technology.

Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, FLAG-tag, Plasmid Preparation, Infection, Control, Virus, Mutagenesis

Journal: Communications Biology

Article Title: TRIM2 inhibits apoptosis by ubiquitinating BNIP3 to protect the intestine against ischemia-reperfusion injury in mice

doi: 10.1038/s42003-025-08708-2

Figure Lengend Snippet: A Caco-2 cells were transfected with either the HA-Vector or HA-TRIM2 plasmid using PEI transfection reagent. The protein expression of TRIM2 and BNIP3 was then detected by western blot. β-actin was used as a loading control (n = 3 biologically independent samples). B Caco-2 cells were transfected with HA-Vector or HA-TRIM2 plasmids using PEI transfection reagent. The relative mRNA level of BNIP3 in the two groups was then detected by qRT-PCR (n = 3 biologically independent samples). C HEK293T cells transfected with varying doses of HA-TRIM2 plasmids were harvested for western blot. β-actin was used as a loading control. D A correlation analysis was conducted to determine the relationship between the protein levels of TRIM2 and BNIP3 in cells ( n = 32 biologically independent samples). ( E ) Caco-2 cells transfected with HA-TRIM2 plasmids were treated with dimethyl sulfoxide (DMSO), MG132 (a proteasome inhibitor), chloroquine (CQ, a lysosome inhibitor), and 3-methyladenine (3-MA, an autophagy inhibitor) for six hours. Western blot was employed to detect the protein expression levels of TRIM2 and BNIP3. The BNIP3 western blot bands were quantified in the different groups ( n = 8 biologically independent samples). β-actin served as a loading control. F HEK293T cells transfected with HA-TRIM2 plasmids were treated with MG132 for 0 h, 1 h, 2 h and 3 h. Western blot was employed to evaluate the protein expression levels of TRIM2 and BNIP3 ( n = 8 biologically independent samples). The quantification of the BNIP3 western blot bands at different time points is presented on the right. β-actin was used as a loading control. G The protein changes of BNIP3 in three groups of Caco-2 cells (transfected with HA-Vector plasmid and treated with DMSO, transfected with HA-TRIM2 plasmid and treated with DMSO, transfected with HA-TRIM2 plasmid and treated with MG132) after CHX treatment for indicated times. The western blot detected the half-life of BNIP3 in three groups, with β-actin serving as a loading control ( n = 6 biologically independent samples). H The protein alterations of BNIP3 in three groups (treated with si-Control, treated with si-TRIM2, treated with si-TRIM2 and HA-TRIM2 plasmid) of Caco-2 cells following CHX treatment for the specified times. The half-life diagram of BNIP3 in the three groups is presented on the right. β-actin was used as a loading control ( n = 9 biologically independent samples). I The protein changes of BNIP3 in three groups (co-transfected with Flag-BNIP3 and HA-Vector, co-transfected with Flag-BNIP3 and HA-TRIM2, and co-transfected with Flag-BNIP3-K130R and HA-TRIM2) of Caco-2 cells after CHX treatment for indicated times. Western blot detected the half-life of Flag-BNIP3 in the three groups ( n = 8 biologically independent samples). β-actin was used as a loading control. J Caco-2 cells were treated with si-BNIP3, and immunofluorescence was performed to detect the co-localization and distribution of TRIM2 and Flag-BNIP3 in the different groups (si-BNIP3+Flag-BNIP3-WT + DMSO, the experimental groups were as follows: si-BNIP3+Flag-BNIP3-WT + MG132, si-BNIP3+Flag-BNIP3-K130R + DMSO, and si-BNIP3+Flag-BNIP3-K130R + MG132. Scale bars are 10 μm. All results are expressed as the mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s test, with the following levels of significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001.

Article Snippet: The BNIP3 antibody (44060S) was purchased from Cell Signaling Technology.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Quantitative RT-PCR, Immunofluorescence

Journal: Communications Biology

Article Title: TRIM2 inhibits apoptosis by ubiquitinating BNIP3 to protect the intestine against ischemia-reperfusion injury in mice

doi: 10.1038/s42003-025-08708-2

Figure Lengend Snippet: A The LDH activity of Caco-2 cell supernatants was evaluated in six experimental groups ( n = 4 biologically independent samples): Control + si-Control, Control + si-TRIM2, Control + si-TRIM2 + si-BNIP3, H/R + si-Control, H/R + si-TRIM2, and H/R + si-TRIM2 + si-BNIP3. B The cell viability of Caco2 was assessed using the CCK-8 assay in the six experimental groups ( n = 6 biologically independent samples). C , D Flow cytometry was employed to ascertain the extent of apoptosis in Caco-2 cells subjected to disparate treatments ( n = 6 biologically independent samples). E Western blot analysis was employed to ascertain the protein expression levels of TRIM2, BNIP3, BAX, Bcl2, BAD and cleaved CASP3 in Caco-2 cells subjected to TRIM2 and BNIP3 protein knockdown and H/R treatment. F The LDH activity of Caco-2 cells in the aforementioned six groups was also evaluated ( n = 4 biologically independent samples). The supernatants of the two cell lines were analyzed in six groups (Control+Empty vector, Control+HA-TRIM2, Control+HA-TRIM2+Flag-BNIP3, H/R+Empty vector, H/R + HA-TRIM2, H/R + HA-TRIM2+Flag-BNIP3). G The cell viability of Caco-2 was determined through the use of a CCK8 assay, which was conducted on six experimental groups ( n = 6 biologically independent samples). H Flow cytometry was employed to ascertain the extent of apoptosis in Caco-2 cells subjected to disparate treatments ( n = 8 biologically independent samples). I Western blot was employed to ascertain the protein expression of TRIM2, BNIP3, BAX, Bcl2, BAD and cleaved CASP3 in Caco-2 cells subjected to TRIM2 and BNIP3 protein overexpression and H/R treatment. J Following transfection of the HA-TRIM2 and Flag-BNIP3 plasmids and subsequent treatment with H/R, Caco-2 cells were stained with JC-1 for 20 minutes at 37 °C. Subsequently, the cells were observed using a laser scanning confocal microscope. Scale bars are 100 μm (Caco-2). K LDH activity was measured in the supernatants of Caco-2 cells in eight groups ( n = 4 biologically independent samples): Control+Flag-BNIP3-WT + HA-Vector, Control+Flag-BNIP3-WT + HA-TRIM2, Control+Flag-BNIP3-K130R + HA-Vector, Control+Flag-BNIP3-K130R + HA-TRIM2. L The cell viability of Caco-2 was assessed using the CCK8 assay in eight groups ( n = 6 biologically independent samples). All results are expressed as the mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s test, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicating statistical significance.

Article Snippet: The BNIP3 antibody (44060S) was purchased from Cell Signaling Technology.

Techniques: Activity Assay, Control, CCK-8 Assay, Flow Cytometry, Western Blot, Expressing, Knockdown, Plasmid Preparation, Over Expression, Transfection, Staining, Microscopy

Journal: Molecular Medicine Reports

Article Title: Nutrient deprivation induces apoptosis of nucleus pulposus cells via activation of the BNIP3/AIF signalling pathway

doi: 10.3892/mmr.2017.7550

Figure Lengend Snippet: ND-induced cell death is associated with increased BNIP3 expression and mitochondrial translocation in nucleus pulposus cells. Cells were subjected to ND for up to 72 h. (A) Protein expression levels of BNIP3 were detected using western blot analysis. (B) BNIP3 protein expression was semi-quantified. (C) Protein extracts of cell fractions from control and treated cells were subjected to western blot analysis with an anti-BNIP3 antibody. (D) BNIP3 expression was normalized as the percentage of the total amount of BNIP3 at 72 h. (E) Dot plot analyses of control and ND incubated with JC-1 and analysed by flow cytometry. Data are presented as the mean ± standard deviation of three experiments. **P<0.01 vs. the vehicle-treated control group. BNIP3, B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein; Cox IV, cytochrome c oxidase IV; Cyto, cytoplasmic; Mito, mitochondrial; ND, nutrient deprivation; Nuc, nuclear.

Article Snippet: Small interfering RNA (siRNA) targeting BNIP3 (cat. no. sc-37452) and AIF (cat. no. sc-29194) along with control siRNA (cat. no. sc-44230) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Translocation Assay, Western Blot, Control, Incubation, Flow Cytometry, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Nutrient deprivation induces apoptosis of nucleus pulposus cells via activation of the BNIP3/AIF signalling pathway

doi: 10.3892/mmr.2017.7550

Figure Lengend Snippet: ND induces nucleus pulposus cell death in a BNIP3-dependent manner. NP cells were transfected with a plasmid expressing BNIP3 or with BNIP3 siRNA. After 24 h, the cells were subjected to ND for a further 72 h. (A and C) BNIP3 expression was detected by western blot analysis. (B and D) Bar graphs presenting the quantification of BNIP3. (E and F) Cell viability was determined by the trypan blue exclusion method. (G and H) Percentage of apoptotic cells was determined using the Annexin V-fluorescein isothiocyanate/propidium iodide assay. Data are presented as the mean ± standard deviation of three experiments. *P<0.05, **P<0.01 vs. the control group; # P<0.05, ## P<0.01 vs. the corresponding ND+control treated group (control siRNA or plasmid). BNIP3, B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein; ND, nutrient deprivation; siRNA, small interfering RNA.

Article Snippet: Small interfering RNA (siRNA) targeting BNIP3 (cat. no. sc-37452) and AIF (cat. no. sc-29194) along with control siRNA (cat. no. sc-44230) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Standard Deviation, Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Nutrient deprivation induces apoptosis of nucleus pulposus cells via activation of the BNIP3/AIF signalling pathway

doi: 10.3892/mmr.2017.7550

Figure Lengend Snippet: AIF serves a key role in BNIP3-induced death of nucleus pulposus cells exposed to ND. Cells were subjected to ND for up to 72 h. (A) Extracts of cell fractions from control and treated cells were subjected to western blot analysis with an anti-AIF antibody. (B) Levels of AIF distribution were normalized as the percentage of the total amount of AIF at each time. NP cells were transfected with either a plasmid expressing BNIP3 or with BNIP3 siRNA. Following 24 h, the cells were subjected to ND for a further 72 h. BNIP3 expression was detected in the (C) plasmid and (D) siRNA groups by western blot analysis. (E) AIF was knocked down by AIF siRNA. At 24 h post-transfection, the cells were subjected to ND for a further 72 h. Cells were collected and lysed, and western blot analysis was performed. (F) Protein expression was semi-quantified for the siRNA groups. (G) Cell viability was determined using the trypan blue exclusion method. (H) Percentage of apoptotic cells was determined using the Annexin V-fluorescein isothiocyanate/propidium iodide assay. Data are presented as the mean ± standard deviation of three experiments. *P<0.05, **P<0.01 vs. the control group; # P<0.05, ## P<0.01 vs. the ND+control siRNA-treated group. AIF, apoptosis-inducing factor; BNIP3, B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein; Cox IV, cytochrome c oxidase IV; Mito, mitochondrial; ND, nutrient deprivation; Nuc, nuclear; siRNA, small interfering RNA.

Article Snippet: Small interfering RNA (siRNA) targeting BNIP3 (cat. no. sc-37452) and AIF (cat. no. sc-29194) along with control siRNA (cat. no. sc-44230) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Control, Western Blot, Transfection, Plasmid Preparation, Expressing, Standard Deviation, Small Interfering RNA

Journal: Physiological reports

Article Title: High-fat diet increases electron transfer flavoprotein synthesis and lipid respiration in skeletal muscle during exercise training in female mice.

doi: 10.14814/phy2.15840

Figure Lengend Snippet: FIGURE 8 Twelve weeks of high-fat diet induced mitochondrial remodeling through fission, fusion, and BNIP3-dependent autophagy and mitophagy mechanisms. (a) markers of mitochondrial dynamics (fission and fusion) and (b) representative blots. (c) whole-cell autophagy and (d) representative blots. (e) markers of mitophagy in mitochondrial subfractions (subsarcolemmal [SSM] and intermyofibrillar [IMFM]) and (f) representative blots. Whole-cell lysates are from quadriceps and isolated mitochondria are from gastrocnemius. Data displayed as mean ± SD. Effects of diet and exercise were evaluated using two-way ANOVA. Images on representative blots come from separate membranes and channels.

Article Snippet: Secondary antibodies were diluted in 5% BSA-TBST or 5% nonfat dry milk-TBST and membranes incubated at room temperature for 1 h. Primary antibodies were diluted 1:1000 and purchased from Abcam (Cambridge, United Kingdom) for OXPHOS cocktail (110413), ETFα (110316), ETFβ (240593), Trimethylated ETFβ (76118), ETFDH (103910), CPT1 (134988), PINK1 (23707), Mitochondrial fission factor (MFF) (81127); Cell signaling (Danvers, MA) for LC3 (12741), p62 (39749), Parkin (4211), and BNIP3 (3769), OPA1 (80471), mitofusion 2 (MFN2) (9482), Abcam, DRP1 (14647); Other primary antibodies included METTL20 (87995, Novus Biologicals, Littleton, CO), HADH (PA5-28203, Thermo Fisher Scientific, Waltham, MA), and BCL2 (7382, Santa Cruz Biotechnology, Dalla, TX).

Techniques: Isolation

Journal: Frontiers in Oncology

Article Title: The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein

doi: 10.3389/fonc.2018.00574

Figure Lengend Snippet: HIF-1 and HIF-2 activation in pRCC and ccRCC samples compared to renal cortex tissue. Representative Western Blots for renal cortex (C) and tumor (T) tissue for pRCC ( n = 41, left panel) and ccRCC ( n = 73, right panel) are shown for HIF-1α and HIF-2α (A,B) , and their downstream targets BNIP3, cyclin D1, Glut-1 and CA9 (C,D) . β-actin was used as a loading control. The example blots in (B) show the same blot probed sequentially after stripping of previous primary and secondary antibodies. Bar charts show the band density of the respective proteins referenced to a control sample (Hypoxia treated T24 cell lysate) which was loaded on each gel. VEGF content of RCC and cortex tissue samples, as measured by ELISA, is reduced in tumor tissue of all grades for pRCC (E) and low grade ccRCC (F) with tendency to be elevated in high grade ccRCC, compared to cortex tissue. Light gray, renal cortex tissue; dark gray, tumor tissue. Statistical significance was assessed with the Wilcoxon matched-pairs signed rank test. Data represent mean + SEM; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, BD610958), HIF-2α (1/400, R&D Systems, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), CA9 (1/200, R&D Systems, AF2188), cyclin D1 (1/10,000, Abcam, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO) as appropriate.

Techniques: Activation Assay, Western Blot, Control, Stripping Membranes, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Oncology

Article Title: The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein

doi: 10.3389/fonc.2018.00574

Figure Lengend Snippet: Spearman correlations between tumor ascorbate content and HIF pathway proteins and HIF pathway scores for pRCC ( n = 41) and ccRCC patients ( n = 73).

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, BD610958), HIF-2α (1/400, R&D Systems, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), CA9 (1/200, R&D Systems, AF2188), cyclin D1 (1/10,000, Abcam, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO) as appropriate.

Techniques:

Journal: Frontiers in Oncology

Article Title: The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein

doi: 10.3389/fonc.2018.00574

Figure Lengend Snippet: Expression of HIF pathway proteins of pRCC and ccRCC tumors in relation to tissue ascorbate content. Bar plots show relative expression values of HIF-1α (A,B) , HIF-2α (C,D) , BNIP3 (E,F) , cyclin D1 (G,H) , GLUT1 (I,J) , CA9 (K,L) , and VEGF (M,N) of pRCC ( n = 41, left panel) and ccRCC tumors ( n = 73, right panel) divided by the respective mean ascorbate content. There was no significant difference between the groups, except for VEGF. However, pRCC tumors with less tissue ascorbate levels tended to have increased HIF pathway protein levels. Differences between the groups where evaluated with the Mann-Whitney test. Data represent mean + SEM; * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, BD610958), HIF-2α (1/400, R&D Systems, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), CA9 (1/200, R&D Systems, AF2188), cyclin D1 (1/10,000, Abcam, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO) as appropriate.

Techniques: Expressing, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein

doi: 10.3389/fonc.2018.00574

Figure Lengend Snippet: HIF pathway expression in ccRCC cell lines in response to ascorbate. Caki-1 (VHL-proficient) and Caki-2 (VHL-defective) cells were pre-loaded with 500 μM ascorbate for 16 h, or left untreated, and then subjected to 21% O 2 or 5% O 2 for 8 h. Protein levels of HIF-1α and BNIP3 were evaluated by Western blot analysis with β-actin as loading control. Densitometry analysis shows protein levels standardized to β-actin and relative to untreated cells (0 μM ascorbate) (A,B) . Induced expression of HIF-1α and BNIP3 in Caki-1 cells at 5% O 2 was repressed by ascorbate treatment. No significant changes in response to ascorbate were observed in Caki-2 cells. Differences between the groups were evaluated by paired t - test. Data represent mean +SD from three independent experiments; * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, BD610958), HIF-2α (1/400, R&D Systems, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), CA9 (1/200, R&D Systems, AF2188), cyclin D1 (1/10,000, Abcam, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO) as appropriate.

Techniques: Expressing, Western Blot, Control

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: Obesity promotes BNIP3-mediated mitophagy in adipose tissue macrophages. (A and B) Uniform manifold approximation and projection (UMAP) plot of SVFs isolated from gonadal white adipose tissue of control mice and mice with obesity (GSE 182,233). Clusters are colored by cell types: tissue-resident macrophages ( Klf4 + ), lipid-associated macrophages ( Trem2 + ), cycling macrophages ( Stmn1 + ), and efferocytes ( Saa3p / Saa3 + ). (C) Schematic diagram of canonical mitophagy pathways. Upon loss of mitochondrial membrane potential, the PINK1-PRKN/Parkin pathway is activated, leading to the recruitment of PRKN to mitochondria and the generation of phospho-ubiquitin (p-ub) chains on outer mitochondrial membrane (OMM) proteins. Adaptor proteins, bind to these p-ub chains and are recognized by LC3. In contrast, hypoxia-induced BNIP3 and BNIP3L/NIX functions independently of PINK1 and PRKN, directly binding to LC3. LC3 then mediates the formation of the phagophore, resulting in the creation of mitophagosomes and the subsequent initiation of mitophagy. (D) Expression levels of Pink1 , Prkn , Bnip3 , and Bnip3l in macrophage subpopulations (as indicated in ) from control mice ( n = 4) and mice with obesity ( n = 4), based on public data analysis (GSE 182,233). (E) Immunofluorescence staining of HIF1A/HIF-1α (green), macrophage marker ADGRE1 (red), and DAPI (blue) in paraffin sections of gWAT of mice fed a NCD or HFD for 12 weeks. Scale bar: 50 μm. (F) qPCR analysis of Pink1 , Prkn , Bnip3 , and Bnip3l in RAW264.7 cells treated with CoCl 2 (100 μM) for the indicated times ( n = 3). (G) Immunoblot analysis of HIF1A and BNIP3 in RAW264.7 cells treated with CoCl 2 (100 μM) for the indicated times ( n = 3). Data are shown as means ± SD. Unpaired t- test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Isolation, Control, Membrane, Ubiquitin Proteomics, Binding Assay, Expressing, Immunofluorescence, Staining, Marker, Western Blot

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: BNIP3 is crucial for CoCl 2 -induced mitophagy in RAW264.7 macrophages. (A and B) Confocal images of RAW264.7 cells expressing mt-Keima, with Bnip3 siRNA knockdown, and treated with CoCl 2 (100 μM) for 6 h ( n = 6). Scrambled siRNA was used as negative control (N.C.) for siRNA knockdown. Mitophagy was quantified by the ratio of red fluorescence (acidic) intensity to green fluorescence (neutral) intensity. Scale bar: 10 μm. (C) Schematic of cas-sgRNA-targeting sites in Bnip3 genome loci. The target site sequence was selected so that the PAM sequence flanks the 3’ end. (D and E) Immunoblot and qPCR analysis to identify the KO of Bnip3 . RAW264.7 cells were transfected with Bnip3 CRISPR-Cas9 KO plasmids or control CRISPR-Cas9 plasmid. (F) immunoblot analysis of mitochondrial proteins in WT and bnip3 KO RAW264.7 cells treated with CoCl 2 (100 μM) for 48 h ( n = 3). (G) Mitochondrial DNA analysis of NC and bnip3 KO RAW264.7 cells treated with CoCl 2 (100 μM) for 48 h ( n = 3). (H and I) Flow cytometry analysis of mitochondrial mass (MitoTracker green) and mitochondrial membrane potential (MitoTracker red) in WT and bnip3 KO RAW264.7 cells treated with CoCl 2 (100 μM) for 48 h ( n = 3). Data are shown as means ± SD. Unpaired t -test or two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Expressing, Knockdown, Negative Control, Fluorescence, Sequencing, Western Blot, Transfection, CRISPR, Control, Plasmid Preparation, Flow Cytometry, Membrane

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: Effects of bnip3 MKO on body weight, tissue weight and energy expenditure in mice with obesity. (A) Body weight monitoring of WT and bnip3 MKO mice during 8 weeks of HFD feeding ( n = 9). (B) Weights of adipose tissue and liver of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 9). (C) Body composition analysis of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 9). (D and E) Hematoxylin and eosin (H&E) staining of gWAT sections of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 6). The average adipocyte size was quantified through adiposoft software using imageJ. Scale bar: 100 μm. (F to K) Indirect calorimetry analysis of WT and bnip3 MKO mice fed a HFD for 12 weeks (EE: energy expenditure; VO 2 : rate of oxygen consumption; VCO 2 : rate of carbon dioxide production; RER: respiratory exchange ratio) ( n = 6). Regression plots of energy expenditure against body mass. ANCOVA test using body weight as a covariate. ancova-adjusted EE was calculated using the pooled slope and mean body weight from regression plots. (L and M) Monitoring of 24 h food intake and locomotor activity of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 6). Data are shown as means ± SD. Unpaired t -test.

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Staining, Software, Activity Assay

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: BNIP3 is essential for obesity-induced mitophagy in adipose tissue macrophages. (A) Immunohistochemical analysis of macrophage marker ADGRE1 (red), mitochondrial marker ACADM (green), and LC3 (red) in paraffin sections of gWAT of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 6). DAPI (blue) was used for nucleus counterstaining. Arrows indicate the colocalization of LC3 and ACADM staining in the merged image. The levels of colocalization between ACADM and LC3 are represented by Pearson’s coefficient, calculated through colocalization analysis using imageJ. Scale bar: 10 μm. (B) Representative TEM of gWAT in WT and bnip3 MKO mice fed a HFD for 12 weeks (ac: adipocytes; Mac: macrophages; black arrow: mitochondria; black arrowhead: autophagosomes). Scale bar: 1 μm (left), 200 nm (right). (C to F) Flow cytometry analysis of mitochondrial mass (MitoTracker green) and mitochondrial membrane potential (MitoTracker red) in SVFs isolated from gWAT of WT and bnip3 MKO mice fed a NCD or HFD for 12 weeks ( n = 3). Macrophage populations were identified by ITGAM + and TREM2 + . Data are shown as means ± SD. Unpaired t -test or two-way ANOVA (** p < 0.01, *** p < 0.001).

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Immunohistochemical staining, Marker, Staining, Flow Cytometry, Membrane, Isolation

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: Macrophage-specific bnip3 KO ameliorates adipose tissue inflammation in mice with obesity. (A) Heat map of the downregulated genes involved in the inflammatory response of bnip3 MKO compared to WT mice fed a NCD or HFD for 12 weeks ( n = 3). (B) qPCR analysis of Tnf , Ccl2 , and Itgax in gWAT of WT and bnip3 MKO mice fed a NCD or HFD for 12 weeks ( n = 7). (C) Immunofluorescence staining of ADGRE1 (green) and DAPI (blue) in paraffin sections of gWAT of WT and bnip3 MKO mice fed a NCD or HFD for 12 weeks. Scale bar: 50 μm. (D and E) Immunoblot analysis ( n = 3) and qPCR analysis ( n = 7) of ADGRE1 in gWAT of WT and bnip3 MKO mice fed a NCD or HFD for 12 weeks. (F to I) Flow cytometry analysis of PTPRC/CD45 + ITGAM + ITGAX/CD11C + cells (M1-like macrophages) and PTPRC + ITGAM + MRC1/CD206 + cells (M2-like macrophages) in SVFs isolated from gWAT of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 6). Data are shown as means ± SD. Two-way ANOVA or unpaired t -test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Immunofluorescence, Staining, Western Blot, Flow Cytometry, Isolation

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: Macrophage-specific bnip3 KO improves glucose tolerance and insulin resistance in mice with obesity. (A) Heat map of the upregulated genes involved in the lipid metabolic process and white fat cell differentiation of bnip3 MKO compared to WT mice fed a NCD or HFD for 12 weeks ( n = 3). (B) qPCR analysis of Slc2a4/Glut4 , Adipoq , and Pparg in gWAT of WT and bnip3 MKO mice fed a NCD or HFD for 12 weeks ( n = 7). (C to F) Intraperitoneal glucose tolerance test (GTT) and intraperitoneal insulin tolerance test (ITT) of WT and bnip3 MKO mice fed a NCD or HFD for 12 weeks ( n = 6). The area under the curve (AUC) measurements are presented. (G to I) Immunoblot analysis of insulin-stimulated AKT phosphorylation in gWAT, liver, and skeletal muscle (quadriceps) of WT and bnip3 MKO mice fed a HFD for 12 weeks ( n = 6). Data are shown as means ± SD. Two-way ANOVA or unpaired t -test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Cell Differentiation, Western Blot, Phospho-proteomics

Journal: Autophagy

Article Title: BNIP3-mediated mitophagy in macrophages regulates obesity-induced adipose tissue metaflammation

doi: 10.1080/15548627.2025.2487035

Figure Lengend Snippet: BNIP3-mediated glycolytic shift is essential for pro-inflammatory activation in RAW264.7 cells. (A to D) Seahorse analysis of oxygen consumption rates (OCR) ( n = 3) and extracellular acidification rate (ECAR) ( n = 5) in WT and bnip3 KO RAW264.7 cells after 24 h of CoCl 2 (100 μM) treatment. (E and F) Immunoblot and qPCR analysis of HIF1A, BNIP3, and IL1B/IL-1β in RAW264.7 cells treated with LPS (100 ng/ml) or CoCl 2 (100 μM) for 24 h ( n = 3). (G and H) Immunoblot and qPCR analysis of HIF1A, BNIP3, and IL1B in RAW264.7 cells treated with 2-DG (1 mm) or CoCl 2 (100 μM) under LPS (100 ng/ml) treatment for 24 h ( n = 3). (I and J) iImmunoblot and qPCR analysis of HIF1A, BNIP3, and IL1B in WT and bnip3 KO RAW264.7 cells treated with CoCl 2 (100 μM) under LPS (100 ng/ml) treatment for 24 h ( n = 3). Data are shown as means ± SD. Unpaired t -test or two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: C57BL/6J- Bnip3 -floxed mice and C57BL/6J- Cx3cr1-Cre mice were purchased from Shanghai Model Organisms Center (Shanghai, China) and Cyagen Biosciences (Suzhou, China), respectively.

Techniques: Activation Assay, Western Blot