Journal: Cell Reports Medicine

Article Title: Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

doi: 10.1016/j.xcrm.2022.100633

Figure Lengend Snippet: Urolithin A confers a proteomic signature of improved mitochondrial metabolism and mitophagy in human skeletal muscle (A) Venn diagram summarizing pathway enrichment analysis results from proteomics data in vastus lateralis skeletal muscle. Data represent upregulated pathways with an adjusted p value <0.1 in subjects treated with placebo or with UA at 500 and 1,000 mg for 4 months compared with baseline. (B and C) Dot plots showing top enriched pathways (WikiPathways 2019 Human), ranked by protein ratio, in the UA 500- (B) and UA 1,000 mg (C) groups from (A). Dot color and size indicate adjusted p value and protein count, respectively. Significant pathways for the placebo group were filtered out to identify treatment-specific pathways. (D) Western blot analysis of protein lysates from vastus lateralis skeletal-muscle biopsies in subjects treated as above. For each subject, both baseline (Pre) and end-of-the-treatment (Post) samples were run, and membranes were probed for phospho-Parkin, total Parkin, and the mitochondrial proteins ATP5A (belonging to the OXPHOS complex V), UQCRC2 (complex IV), SDHB (complex II), and NDUFB8 (complex I). Tubulin and VDAC were included as markers of total and mitochondrial protein abundance, respectively. Dashed line separates samples from individual subjects. (n = 6 Pre and Post, biologically independent samples). (E) Quantification of phospho-Parkin over Tubulin protein intensity from western blots (WBs) in (D) (n = 6). Two-sided, paired t-test. (F) Quantification of NDUFB8 (left) and SDHB (right) protein intensity, normalized over VDAC from WBs in (D) (n = 6). ∗p < 0.05; ∗∗p < 0.01; two-sided, paired t test.

Article Snippet: The following primary antibodies were incubated overnight diluted in blocking buffer: UBE2N (SantaCruz, sc-376470, 1:3000), Tubulin (Proteintech, 10004185, 1:3000), phospho-Parkin S65 (Cell Signaling, #36866, 1:1000), Parkin (Cell Signaling, #4211, 1:1000), OXPHOS Antibody Cocktail (Abcam, ab110413, 1:2000), VDAC (Proteintech, 10866-1-AP, 1:1000).

Techniques: Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

doi: 10.1016/j.xcrm.2022.100633

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were incubated overnight diluted in blocking buffer: UBE2N (SantaCruz, sc-376470, 1:3000), Tubulin (Proteintech, 10004185, 1:3000), phospho-Parkin S65 (Cell Signaling, #36866, 1:1000), Parkin (Cell Signaling, #4211, 1:1000), OXPHOS Antibody Cocktail (Abcam, ab110413, 1:2000), VDAC (Proteintech, 10866-1-AP, 1:1000).

Techniques: Recombinant, RNA Extraction, Software, Protease Inhibitor

Journal: Aging Cell

Article Title: The ultrastructural and proteomic analysis of mitochondria‐associated endoplasmic reticulum membrane in the midbrain of a Parkinson's disease mouse model

doi: 10.1111/acel.14436

Figure Lengend Snippet: Initial proteomic parsing of midbrain MAM fractions in controls and MPTP‐treated mice. (a) Subcellular fractions extracted from midbrain tissues were validated by specific organelle protein markers. Calnexin, Calreticulin, and Sigma‐1R were considered as consensus proteins for ER and MAM. VDAC1 and Cox IV were applied for mitochondria and MAM markers, and GAPDH was to prove cytosolic fractions. (b) Venn graph of identified proteins in each group ( n = 5 per group) and consensus MAM proteins. (c, d) Sample distribution plots by PCA (c) and PLS‐DA analysis (d) in MAM proteomics between controls and MPTP‐treated mice.

Article Snippet: Primary antibodies used in this study included that anti‐tyrosine hydroxylase (TH) rabbit mAb (1:2000, Cat. 58844, Cell Signaling Technology), anti‐glial fibrillary acidic portein (GFAP) rabbit mAb (1:2000, Cat. 80788, Cell Signaling Technology), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) rabbit mAb (1:2000, Cat. 5174, Cell Signaling Technology), anti‐cytochrome‐c‐oxidase subunit 4 (Cox IV) rabbit Ab (1:2000, Cat. 5844, Cell Signaling Technology), anti‐sigma‐1 receptor (Sig‐1R) rabbit mAb (1:2000, Cat. 61994, Cell Signaling Technology), anti‐phosphatase and tensin homolog (PTEN) rabbit mAb (1:2000, Cat. 9188, Cell Signaling Technology), anti‐calreticulin rabbit mAb (1:2000, Cat. 92516, Abcam), anti‐calnexin (CNX) rabbit pAb (1:2000, Cat. ADI‐SPA‐860, Enzo Life Sciences), anti‐voltage dependent anion channel 1 (VDAC1) rabbit pAb (1:2000, Cat. 55259‐1‐AP, Proteintech), anti‐leucine‐rich repeat kinase 2 (LRRK2) rabbit mAb (1:1000, Cat. R380823, Zen‐bioscience), anti‐excitatory amino acid transporter 2 (EAAT2) rabbit mAb (1:1000, Cat. R381853, Zen‐bioscience), anti‐tropomyosin‐1 (TPM1) rabbit mAb (1:1000, Cat. R383145, Zen‐bioscience), and anti‐coatomer protein complex subunit zeta 1 (COPZ1) rabbit pAb (1:1000, Cat. 824631, Zen‐bioscience).

Techniques:

Journal: BMC Molecular and Cell Biology

Article Title: Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

doi: 10.1186/s12860-020-00288-9

Figure Lengend Snippet: Western blotting and densitometric analysis of LRP-1, thrombospondin, VDAC1, VDAC2 and annexin A1 in cell lysates of chondrocytes under inflammatory (IL-1β + TNF-α, both 10 ng/mL) versus control conditions. a Non-significant decrease in LRP-1 expression upon cytokine exposure. b Increase in thrombospondin expression upon cytokine exposure. c Similar VDAC1 expression. d Similar VDAC2 expression. e Annexin A1 expression showed a trend to decrease upon cytokine exposure. f Beta-actin was measured on each western blot separately as a loading control to correct for the exact amount of protein per lane. Measurements from three horses (three biological replicates) were combined to provide final values for each group (mean ± SD)

Article Snippet: VDAC1 (C-terminal) , Aviva Systems Biology , ARP35122 (polyclonal) , 1:250 , IRDye 680RD Goat , LI-COR Biosciences , 926–68,071 , 1:5000.

Techniques: Western Blot, Control, Expressing

Journal: BMC Molecular and Cell Biology

Article Title: Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

doi: 10.1186/s12860-020-00288-9

Figure Lengend Snippet: Western blotting and densitometric analysis of VDAC1 and VDAC2 monomers and dimers in cell lysates of chondrocytes upon cytokine exposure (IL-1β + TNF-α, both 10 ng/mL) versus control conditions. a VDAC1 monomer expression was significantly decreased upon cytokine exposure. VDAC1 dimer expression was unchanged. b Similar VDAC2 monomer and dimer expression. Measurements from three horses (three biological replicates) were combined to provide final values for each group (mean ± SD)

Article Snippet: VDAC1 (C-terminal) , Aviva Systems Biology , ARP35122 (polyclonal) , 1:250 , IRDye 680RD Goat , LI-COR Biosciences , 926–68,071 , 1:5000.

Techniques: Western Blot, Control, Expressing

Journal: BMC Molecular and Cell Biology

Article Title: Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

doi: 10.1186/s12860-020-00288-9

Figure Lengend Snippet: Detailed specification of primary and secondary antibodies employed for western blotting

Article Snippet: VDAC1 (C-terminal) , Aviva Systems Biology , ARP35122 (polyclonal) , 1:250 , IRDye 680RD Goat , LI-COR Biosciences , 926–68,071 , 1:5000.

Techniques: Western Blot

Journal: bioRxiv

Article Title: SIRT3 deficiency decreases oxidative-metabolism capacity but increases lifespan under caloric restriction

doi: 10.1101/2022.05.09.491205

Figure Lengend Snippet: A) Immunoblot of pan-acetylation, normalized to VDAC, for liver mitochondrial enrichment from 25-month-old treatment groups. Data were analyzed by two-way ANOVA followed by multiple comparisons test. p value reported for each comparison is corrected by Tukey’s test. Results are plotted as mean ± SEM. *: p≤0.05. B) Percentage of significantly changed acetyl-lysine residues that show increased stoichiometry due to Sirt3 -/- , calculated by (the number of acetyl-lysine sites showing increased stoichiometry) / (the number of significantly changed acetyl-lysine sites, p<0.05) x100%. C) Heat map of significantly changed lysine sites (p<0.1) that are response to loss of SIRT3. Plotted sites are significantly changed (p<0.1) in either Sirt3 -/- CD vs. WTCD or Sirt3 -/- CR vs. WTCR comparison. Values are colored based on relative acetylation stoichiometry, normalized to the median value of each sites in all four groups, scaling ranging from -0.8 to 0.8 (x100%). D) Functional cluster analysis of KEGG pathways (DAVID 6.8). Significantly enriched (-log10(p value) >1.5) pathways are indicated, with Sirt3 -/- CD vs. WTCD in orange and Sirt3 -/- CR vs. WTCR in blue. E) Acetylation sites in FAO and BACC metabolism, TCA cycle, and ETC that displayed larger than 5% stoichiometry (p<0.1) for Sirt3 -/- CD vs. WTCD (orange colored) and Sirt3 -/- CR vs. WTCR comparison (blue colored).

Article Snippet: Western blot primary antibodies used include: SIRT3 (#5490, CST, 1:1000), Acetylated-Lysine (#9681, CST, 1:1000), VDAC (75-204, NeuroMab, 1:1250).

Techniques: Western Blot, Comparison, Functional Assay

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 4. Biophysical validation reveals VDAC1 as a new protein target of Sert. (A) The DARTS assay for target validation. VDAC1 protein stability was increased upon Sert (100 μM) treatment in HUVECs lysates. Pronase treatment was conducted for 5, 10, and 20 min. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05, **p < 0.01. (B) The DARTS assay demonstrated the dose-dependent binding of Sert to VDAC1. Treatment with pronase (5 μg/mL) was conducted for 10 min. The graph plots the quantification data is shown in (B). n = 3, *p < 0.05, **p < 0.01. (C) In silico docking model of Sert interacting with VDAC1 (human VDAC1, RCSB PDB ID: 2JK4). Sert bound to the VDAC1 between α-helix and β-sheet in the most stable pose and binding motifs were depicted with several high- affinity interactions between Sert and the VDAC1 pocket. Ligands are shown as gray sticks in displayed hydrogen bond surfaces. Bonds are shown as dashed lines color-coded as follows: hydrophobic interactions in orange, electrostatic interaction in purple, and hydrogen bonds in green and sky blue.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Biomarker Discovery, Binding Assay, In Silico

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 5. Biological validation using vdac1-knockout cells reveals that VDAC1 is a biological relevant protein target of Sert. (A) Western blot analysis of the levels of VDAC1, p-MTOR, MTOR, p-AMPK, AMPK, and ACTB in cells after each compound treatment for 30 min in WT MEFs or vdac1-knockout MEFs. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05, **p < 0.01. (B) MAP1LC3 positive puncta (green) were increased by Sert (5 μM) treatment in WT MEFs, whereas vdac1-knockout MEFs treated with Sert did not induce autophagic vesicles formation. The cells were treated with Sert (5 μM) or Rapa (10 μM) for 24 h in HUVECs. And then, the cells were harvested, followed by treatment with MAP1LC3B (green) antibody for 1 h. Nuclei were stained with DAPI. Images were obtained from confocal microscopy. Quantification of data is shown in (B). Values are means ± SEM; n > 10 cells, ***p < 0.001; scale bar: 10 μm. (C) Autophagic flux evaluation in WT MEFs or vdac1-knockout MEFs using mCherry-GFP MAP1LC3 in the presence of each compound. Representative images of merged channels are shown; scale bar: 10 μm. Pearson coefficient for the colocalization analysis is shown. Quantification of data is shown in (C). Values are means ± SEM; n > 10 cells, **p < 0.01, ***p < 0.001. (D) Effect of Sert treatment on the proliferation of WT MEFs and vdac1-knockout MEFs. All cells were treated with Sert (0–5 μM) for 72 h, and cell growth was measured using an MTT colorimetric assay. N = 3, Values are means ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Biomarker Discovery, Knock-Out, Western Blot, Staining, Confocal Microscopy, Colorimetric Assay

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 6. Validation of the binding site of Sert on VDAC1 (A) For DARTS assay, vdac1−/- MEF cells were transfected with WT MYC-VDAC1, MYC-VDAC1H184P or MYC- VDAC1S196A vectors at 1500 nM each for 48 h. And then, the cells were harvested, lysed, followed by treatment with (Sert, 100 μM) treatment. Pronase treatment (5 μg/mL) was conducted for 10 min. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05. (B) VDAC1−/- MEF cells were transfected with WT MYC- VDAC1, MYC-VDAC1H184P or MYC-VDAC1S196A vectors at 200 nM each for 24 h. Then, the cells were treated with Sert (5 μM) for an additional 24 h. And then, the cells were harvested, followed by treatment with MYC (red) and MAP1LC3B (green) antibody for 3 h. Nuclei were stained with DAPI. Images were obtained from confocal microscopy. Quantification of data is shown in (B). Values are means ± SEM; n > 10 cells, *p < 0.05, **p < 0.01, ***p < 0.001; scale bar: 10 μm.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Biomarker Discovery, Binding Assay, Transfection, Staining, Confocal Microscopy

Journal: Autophagy

Article Title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.

doi: 10.1080/15548627.2020.1841953

Figure Lengend Snippet: Figure 9. VDAC1 is a biologically relevant target protein of Sert-induced biological activity on tauopathy. (A) Western blot analysis of the level of VDAC1, p-MAPT, MAPT, Cleaved MAPT, MAP1LC3, and ACTB in inducible MAPT cells after transfection with si-VDAC1 (100 nM) for 48 h. The graph plots the quantification data is shown in (A). n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Degradation of MAPT oligomers was visualized in MAPT-BiFC cells when cells were transfected with si- VDAC1 (100 nM) for 48 h. Scale bar: 20 μm. Quantification of data is shown in (B). Values are means ± SEM; n > 20 cells. ***p < 0.001. (C) Schematic summary of VDAC1-mediated autophagy modulation upon Sert treatment.

Article Snippet: Recombinant VDAC1 plasmid transfection analysis For transient overexpression of VDAC1, MYC-VDAC1 (Origene, RC209949) was purchased.

Techniques: Activity Assay, Western Blot, Transfection

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: SMR discovery and experimental validation of NETs-related biomarkers in osteoporosis. A, Forest plot summarizing SMR results for VDAC1, PLCG2 and NCF2 (β, SE, P, HEIDI P, and OR ± 95% CI). B, Representative IHC images of distal femur (brown = positive DAB; hematoxylin counter-stain) showing increased staining for the three proteins in OVX rats versus Sham; scale bar = 50 µm. C, Western blots of neutrophil lysates (Control vs PMA-induced NETs) probed for NCF2 (65 kDa), VDAC1 (31 kDa) and PLCG2 (148 kDa); GAPDH (36 kDa) serves as loading control. D–F, Quantification of IHC-positive area (% of marrow) for each protein (mean ± SD). G–I, Densitometric ratios of target proteins to GAPDH in western blots (mean ± SD). P < 0.05*, < 0.01 **, < 0.001 ***, < 0.0001 **** (two-tailed t-test).

Article Snippet: The sections were incubated in a blocking solution to reduce non-specific binding, followed by overnight incubation at 4°C with primary antibodies: VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), and NCF2 (Novus Biologicals, NBP3-16257).

Techniques: Biomarker Discovery, Staining, Western Blot, Control, Two Tailed Test

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Docking results of available proteins small molecules. (A) NCF2 docking Diltiazem. (B) VDAC1 docking Diltiazem. (C) PLCG2 docking Exemestane. (D) NCF2 docking Cimetidine. (E) VDAC1 docking Cimetidine. (F) NCF2 docking Desipramine. (G) NCF2 docking Profenamine. (H) VDAC1 docking Emodin. (I) VDAC1 docking Tolazoline. (J) VDAC1 docking Zuclopenthixol. (K) VDAC1 docking Ethynyl estradiol. (L) PLCG2 docking Simvastatin.

Article Snippet: The sections were incubated in a blocking solution to reduce non-specific binding, followed by overnight incubation at 4°C with primary antibodies: VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), and NCF2 (Novus Biologicals, NBP3-16257).

Techniques: