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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

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: The SiRNA (vdac1‐homo‐815, 19601) and plasmid (HG17755‐NF, Sino Biological) were transfected for knockdown and overexpression of VDAC1, respectively; 0.25 mmol/L DIDS (32029, MCE) was used to inhibit VDAC1.

Techniques: Construct, Expressing

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: International Journal of Molecular Sciences

Article Title: Integrated Multilayer Omics Reveals the Underlying Mechanisms in Xylazine-Related Heart Injury in Rats

doi: 10.3390/ijms26178532

Figure Lengend Snippet: Validation of related proteins in the fructose and mannose metabolism signaling pathway and cholesterol metabolism signaling pathway. ( A ) Western blot image of VDAC3. ( B ) Quantification of VDAC3. ( C ) Western blot image of PFKM. ( D ) Quantification of PFKM. * p < 0.05 compared with the control group.

Article Snippet: The membranes were incubated in 5% skimmed milk for 2 h, and then in diluted primary antibodies against PFKM (1:2000, 55028-1-AP, Proteintech, Rosemont, IL, USA); VDAC3(1:500, 55260-1-AP, Proteintech), GAPDH (1:10,000, 60004-1-Ig, Proteintech), tubulin (1:10,000, GB15140-100, Servicebio) at 4 °C 12 h. They were incubated in Goat Anti-Rabbit IgG H&L/HRP (1:10,000, GB23303, Servicebio) or Goat Anti-Mouse lgG H&L/HRP (1:10,000, GB23301, Servicebio) for 1 h. A chemiluminescence instrument was used to image the bands and Image J v1.8.0.345 was used to calculate the gray values.

Techniques: Biomarker Discovery, Western Blot, Control

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: In Vitro, Activity Assay, Staining, Western Blot, Two Tailed Test, Quantitative RT-PCR, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: TGF-β signaling in CCN2/CTGF treated HTM-N cells in vitro and in βB1-CTGF1 mice in vivo . (A) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. mRNA expression of Tgf-b1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 8, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 3). Tgf-b2 mRNA was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 5 ng/mL CCN2/CTGF: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 5). mRNA expression of Ccn2/Ctgf was significantly increased after the treatment with 5 ng/mL and 50 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 6, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 6). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (B) Western blot analysis of TGF-β1 and TGF-β2 in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. Protein synthesis of TGF-β1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (n = 4). The protein synthesis of TGF-β2 was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 5). Right panel shows representative Western Blots for the proteins. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (C) Real-time RT-PCR analysis of Tgf-b1 and Tgf-b2 in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. mRNA expression of Tgf-b2 was significantly increased in βB1-CTGF mice, compared to wildtype littermates ( Tgf-b1 : WT: n = 8, TG: n = 6; Tgf-b2 : WT: n = 19, TG: n = 19). For statistical analysis the Mann-Whitney test was used. mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. (D) Western blot analysis of TGF-β2 in the anterior eye segment of 2-month-old βB1-CTGF mice and wildtype littermates. Protein synthesis of TGF-β2 was significantly increased in βB1-CTGF1 mice, compared to wildtype littermates (TGF-β2: WT: n = 6, TG: n = 6). Right panel shows a representative Western blot. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (E) Immunohistochemical staining of TGF-β2 (red) in the anterior chamber of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of TGF-β2 was increased in the TM of βB1-CTGF mice1, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). n = 5 (F) Western blot analysis of the phosphorylation of Smad2 and Smad3. Protein synthesis of pSmad2 and pSmad3 was significantly increased in βB1-CTGF mice, compared to wildtype littermates (pSMAD2: WT: n = 6, TG: n = 6; pSmad3: WT: n = 7, TG: n = 7). Right panel shows representative Western Blots for both proteins. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (G) Immunohistochemical staining of pSmad2 (red) in the anterior chamber angle of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of pSmad2 is increased in the TM of βB1-CTGF1 mice, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). Data represented as mean ± SD. CB: ciliary body, I: iris, C: cornea, TM: trabecular meshwork. n = 5. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: CCN2/CTGF induced TGF-β signaling activation is mediated via the Erk- and RhoA/ROCK signaling pathway. (A) Real-time RT-PCR analysis of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the inhibition of the Erk-pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Mek1/2 inhibitor only, compared to DMSO treated control cells (n ≥ 4; # p ≤ .05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (B) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n ≥ 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. (C) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. (D) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf in HTM-N cells after the inhibition of the RhoA/ROCK signaling pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Fasudil only, compared to DMSO treated control cells (n ≥ 5; # p ≤ 0.05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (E) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 3, # p ≤ 0.05 to DMSO control; ** p < 0.01 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set to1. α -Tubulin was used to normalize protein synthesis. (F) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 was significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 7, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. Dotted line indicates the control group treated with the solvent of the inhibitor for the appropriate experiment. For statistical analysis a one-way ANOVA was performed.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: Activation Assay, Quantitative RT-PCR, Inhibition, Expressing, Western Blot, Staining

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: KEGG pathway clustering analysis of different comparison groups, the PPI network of differentially lactylated proteins in the pathways of interest, and verification of Vdac3 protein lactylation modifications. (A) Heatmap of lactylation sites for 12 key proteins. Each row represents a differentially modification site; each column represents a sample. Red indicates a high-level modification, blue indicates a low-level modification, and grey indicates sites that cannot be quantified in the corresponding samples. (B) The horizontal axis depicts different comparison groups, whereas the vertical axis indicates the enriched KEGG pathways. The colored blocks represent the functional descriptions of differentially expressed modified proteins enriched in each comparison group. Blue signifies high enrichment significance, whereas blue-white signifies low enrichment significance. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) PPI network of differentially lactylated proteins involved in the 4-VO group and sham group in the five pathways of interest. (D) PPI network in the 4-VO group and 4-VO + EA group. The color of the outer circle indicates the corresponding KEGG pathway of the protein, whereas the shape within the circle denotes the upregulation or downregulation of the modification sites contained within the protein. A diamond shape signifies proteins with upregulated modification sites, a downward arrow indicates proteins with downregulated modification sites, and a circle signifies proteins with upregulated and downregulated modification sites. The red box indicates the Vdac3 protein. (E) The IP experiment verified the lactylation modification level of Vdac3. (F) Quantification was performed using ImageJ software, and the values are expressed as the mean with the corresponding standard error from three replicate experiments ( n = 3, compared with the sham group, ## p < 0.01; compared with the 4-VO group, ** p < 0.01). 4-VO, four-vessel occlusion; EA, electroacupuncture; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction; IP, immunoprecipitation; Vdac3, mitochondrial voltage-dependent anion channel protein3.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Comparison, Modification, Functional Assay, Software, Immunoprecipitation

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: Analysis of lactylation site motifs in different groups and representative mass spectra of key proteins. (A) A heatmap of amino acids adjacent to lysine lactylation sites, with a green/red scale (−5 to 5) indicating the frequency of amino acid detection; red indicates high frequency; green indicates low frequency. The height ratio of amino acid abbreviation letters at specific positions reflects motif characteristics, with a % difference greater than 0 indicating a higher frequency of that amino acid at that position compared to the background, and less than 0 indicating a lower frequency. (B) Four conserved motif sites of lysine. (C) Sequence motif map of lactylation modification, 10 amino acids upstream and downstream of the modification site were selected, and the vertical axis indicates the difference between the proportion of the various amino acids at the same site in the experimental data set and the reference background, that is, percentage difference (%difference). The height ratio of amino acid abbreviation letters at specific positions represents the motif characteristics, and a %difference greater than 0 indicates that the frequency of the amino acid at this position is higher than the background. Values lower than 0 indicate that the frequency of the amino acid at this position is lower than the background. (D–F) The lactylation site of Vdac3 was identified by mass spectrometry. PPI, protein–protein interaction; 4-VO, four-vessel occlusion; EA, electroacupuncture.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Sequencing, Modification, Mass Spectrometry