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: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: VDAC1 is dysregulated in NAFLD‐driven HCC and associated with NAFLD. A, Volcano plot graph showing the differential expressed proteins in the quantitative analysis. The −log 10 ( P ‐value) was plotted against the log2 (ratio Tumor/Normal). The upregulated proteins in HCC tissues were marked with red dots, and the downregulated proteins in HCC tissues with green dots. B, Pathways analysis of significantly altered proteins. The canonical pathways associated with the differential proteins were tested alongside the P ‐values calculated using right‐tailed Fisher exact test. The top 10 pathways were listed. C, Transcriptome‐wide association study suggested that VDAC1 was associated with NAFLD. The correlation between differentially expressed protein and the phenotype of body fat mass percentage (Record ID 12 919) was shown. The red plots indicated the positive correlation, the green plots indicated the negative correlation, and the dark plots indicated no statistical significance. A significant correlation with the VDAC1 ( R = 0.469, P = .008) was shown in the red triangle. D, Overexpression of VDAC1 in tumor tissues was validated with western blot. C represented the HCC tissues, and N represented the adjacent normal tissues. RPLP0 is used as internal reference. E, Heat map showed that VDAC1 was associated with a serial of fatty liver disease phenotypes in both gene and protein expression level. The VDAC1 transcript had significant correlations with the body fat mass ( R = 0.472, P = .008), body weight ( R = 0.361, P = .049), liver mass ( R = 0.358, P = .021), white adipose mass ( R = 0.391, P = .035), cholesterol in plasma ( R = 0.525, P = .0003), Glucose AUC in OGTT ( R = 0.331, P = .034) and fasting glucose level ( R = 0.298, P = .058). Red represents positive correlations, and blue represents negative correlations. Transcript and peptide 1‐20 represent the gene and peptides expression level, respectively. A, B, D, obtained from the proteomic analysis of human samples; C, E were obtained from the transcriptomic and proteomic analysis of BXD family samples

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Over Expression, Western Blot, Expressing, Clinical Proteomics

Journal: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: Identification of VDAC1 expression correlated with HCC in a human clinical cohort. A, Vdac1 upregulated in HCC samples compared with the normal samples was validated in the Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA‐LIHC) ( n = 371), and Normal ( n = 50) cohorts. B, Vdac1 is upregulated with HCC grade increase and was identified in TCGA‐LIHC Grade 1 ( n = 54), Grade 2 ( n = 173), Grade 3 ( n = 118), Grade 4 ( n = 12), and Normal ( n = 50) cohorts. C, High‐level VDAC1 expression correlates with poor survival outcome in HCC and LIHC (High, n = 91; Low, n = 91) cohorts based on survival analysis

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Expressing

Journal: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: System genetics analysis of Vdac1 in BXD mice strains. A, Variable expression levels of Vdac1 in the liver tissue. Expression data for 39 BXD strains and their parental strains (B6 and D2). The x‐axis denotes the strain, while the y‐axis denotes mean expression given as log 2 . Each bar shows mean expression values ± standard error of the mean (SEM). B, Interval mapping of the Vdac1 gene in the liver. The upper x‐axis shows the chromosome, the lower x‐axis shows the location in megabases, the y‐axis provides the likelihood ratio statistics score (LRS). Blue lines indicate the LRS values at a given position, with a significant LRS at 18.35 and suggestive LRS at 11.14. Interval mapping identified a significant cis ‐eQTL on chromosome 11 at 52.01 Mb and 4 suggestive trans ‐eQTLs on chromosomes 2, 5, 14, and X

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Expressing

Journal: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: List of 4 suggested trans ‐eQTLs for Vdac1

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Marker

Journal: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: VDAC1 is associated with mitochondria dysfunction. A, Vdac1 co‐expression network is involved in mitochondrial dysfunction based on the mice liver transcriptome data. The Vdac1 co‐expression genes were analyzed by IPA. The top canonical pathways associated with the network genes were tested alongside the P ‐values calculated using a right‐tailed Fisher exact test. Mitochondrial dysfunction was listed as the top pathway. B, Vdac1 shows correlation with 20 mitochondrial genes. The red plots indicate the significant positive correlation, and the dark plots indicate no statistical significance. C, Heatmap showing the correlation between VDAC1 and different species of CL based on the BXD family database. VDAC1 transcript had a negative relationship with the predominant CL species, CL (LLLL) and its precursor MLCL (LLL). VDAC1 had a positive relation with the nascent CL; and the VDAC1 correlated peptides showed the same trend. Red represents positive correlations, and blue represents negative correlations. D, Correlation between Vdac1 and cardiolipin synthetase gene Ptpmt1 and Tafazzin in a BXD mice cohort. Vdac1 has a positive correlation with Ptpmt1 ( R = 0.452, P = .003); and negative correlation with Tafazzin ( R =−0.334, P = .033) among 41 BXD strains. The blue line represents the correlation with Ptpmt1 , and green with Tafazzin

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Expressing

Journal: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: Mitochondrial function analysis with or without VDAC1 expression in Hep3B and HepG2. A, Western blot analysis of VDAC1 level in HepG2 cells after transfection of VDAC1 siRNA and VDAC1 expression plasmid. Compared with the negative control, the expression level of VDAC1 reduced in the knockdown group, and increased in overexpression group. RPLPO was the internal reference protein. B, Determination of cardiolipin in Hep3B and HepG2 cells. Cardiolipin was measured using the fluorometric probe assay kit (ab241036). The left panel shows the cardiolipin content in Hep3B. Compared with the NC group, the cardiolipin contents both in knockdown group and inhibitor group were significantly higher ( P < .01), and the cardiolipin content in overexpression group was lower ( P < .05). The right panel showed the cardiolipin content in HepG2. Compared with the NC group, the cardiolipin contents both in knockdown group and inhibitor group were significantly higher ( P < .01), and the cardiolipin content in overexpression group was lower ( P < .05). C, Detection of cardiolipin levels in HepG2 and Hep3B by NAO staining. Compared with negative control, the fluorescence intensity increased in the knockdown group and inhibitor group, and decreased in the overexpression group both in Hep3B and HepG2. The fluorescence intensity of the 2 cell lines was observed under a microscope at ×10 and ×40 magnification, respectively. D, Detection of mitochondrial respiratory function in Hep3B and HepG2 cells. High‐resolution respirometry performed in an Oroboros Oxygraph‐2k. The different color curves showed the mitochondrial oxygen consumption of the different groups. In Hep3B cells, the OCR of knockdown group and inhibitor group were higher than the NC group, and the OCR of overexpression group and inhibitor group was lower than the NC group. Also, the same trend was shown in HepG2 cells. E, Relative VDAC1 expression was detected by IHC between NAFLD‐driven HCC and adjacent non‐tumor liver tissues (×40). N represented the VDAC1 in adjacent non‐tumor liver tissues. T represented the VDAC1 in NAFLD‐driven HCC tissues. (NC represented the negative control group; VDAC1 siRNA represented VDAC1 knockdown group; VDAC1 represented the overexpression group and DIDS represented the inhibitor group)

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Negative Control, Knockdown, Over Expression, Staining, Fluorescence, Microscopy

Journal: Cancer Science

Article Title: System biology analysis reveals the role of voltage‐dependent anion channel in mitochondrial dysfunction during non‐alcoholic fatty liver disease progression into hepatocellular carcinoma

doi: 10.1111/cas.14651

Figure Lengend Snippet: VDAC1‐centralized correlation network. A, Correlation network of VDAC1‐centralized transcript‐lipids phenotypes based on the BXD mice family database. The network was constructed including VDAC1 expression, NAFLD phenotypes and representative CL species. Red lines show positive correlations, and blue lines show negative correlations. The network illustrates the VDAC1 associated with NAFLD phenotypes through correlating with CL profiling. B, Schematic for VDAC1 involved in NAFLD‐related HCC. VADC1 is upregulated in NAFLD, and dysregulation of VDAC1 causes a CL acyl chain composition shift, which led the NAFLD‐driven HCC tumorigenesis through mitochondrial dysfunction

Article Snippet: After treated with 3% hydrogen peroxide for 30 min, the samples were blocked with bovine serum albumin for 30 min at 37°C, and incubated in the VDAC1 antibody (CSB‐RA025821A0HU, CUSABIO, 1:100), then followed by the Histostain‐SP (streptavidin‐peroxidase) kit (SP‐0023, BIOSS) protocol.

Techniques: Construct, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pink1/PARK2/mROS-Dependent Mitophagy Initiates the Sensitization of Cancer Cells to Radiation

doi: 10.1155/2021/5595652

Figure Lengend Snippet: Mitochondrial dysfunction caused by mROS from Sarm1-KR and IR. (a) Representative FCM pictures of MMP in HeLa cells stained by Rh123 and analyzed by FCM. (b) The changes of Na + K + -ATPases, Ca 2+ Mg 2+ -ATPase, and mito-respiratory complex I and III activities by biochemical assay after light exposure and IR. (c) WB analysis was performed to determine protein levels of VDAC1 in total- and mito-protein. β -Actin and HSP60 proteins were used for loading control. (d) From top to bottom, the changes of VDAC1/ β -actin and VDAC1/HSP60. Bars represent mean ± SD of triplicate measurements. ∗ P < 0.05 and ∗∗ P < 0.01, versus control; # P < 0.05, versus 4 Gy IR; and △ P < 0.05, versus light exposure.

Article Snippet: Anti-COX IV, anti- β -actin, and anti-GAPDH were purchased from Santa Cruz, CA, USA; anti-voltage-dependent anion channel 1 (VDAC1), anti-heat-shock protein 60 (HSP60), anti-Pink1, and anti-PARK2 were purchased from Bioworld Technology, Inc., USA; and anti-microtubule-associated protein 1 light chain 3 (LC3), anti-p62, and anti-Tom20 were purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Staining

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: Pathway enrichment.

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Expressing

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: Immunoblotting analysis to validate the differential expression for 9 proteins in VPA-treated and PBS-treated biological replicated SH-SY5Y cells. ( A ) Immunoblotting showed the expression of ATP5J, FBXO4, HDAC1, HDAC2, SMARCA4, UBE1L, UBE2D1, UQCRB, and VDAC17 in VPA-treated and PBS-treated SH-SY5Y cells. ( B ) Quantification showing the fold differences in the expression of ATP5J, FBXO4, HDAC1, HDAC2, SMARCA4, UBE2D1, UQCRB, and VDAC1 between VPA-treated groups and PBS control. GAPDH was a loading control. The data are expressed as fold change to PBS ± SD (* p < 0.05, n = 3 to 6).

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Western Blot, Expressing

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: RT-qPCR assay showing the expression of 14 genes ( ATP5F1A , ATP5PF , BAZ1B , BMS1 , FBXO2 , HDAC1 , HDAC2 , SDHA , SMARCA4 , UBA7 , UBE2D1 , UQCRB , VDAC1 , and WDR36 ) in VPA-treated and PBS-treated SH-SY5Y cells. The GAPDH gene was used as the endogenous gene for normalization. The data are expressed as fold change to PBS ± SD (* p < 0.05, n = 3 to 6).

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Quantitative RT-PCR, Expressing