Journal: Molecular Cancer

Article Title: Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex

doi: 10.1186/1476-4598-10-62

Figure Lengend Snippet: Exogenous GABPβ in SK-BR-3 cells restores BRCA1 proximal promoter activity and stabilizes endogenous GABPα . ( a ) Expression vectors for the individual GABP subunits, or both together, were cotransfected with the BRCA1 L6-pRL promoter construct in SK-BR-3 cells. ( b ) SK-BR-3 cells were co-transfected with the indicated FLAG-tagged GABP expression vectors. Whole cell lysates from these cells were analyzed by Western blots probed with antibodies against GABPα, GABPβ or the FLAG moiety and then reprobed with anti-γ-tubulin to control for sample loading. The arrow indicates the band corresponding to endogenous GABPα protein. Apparent molecular weight markers (kDa) are presented to the right of the panels.

Article Snippet: The final FLAG-GABPβ construct was generated by the ligation of these two fragments. pTRE-tight-GABPβ was prepared by digesting FLAG-GABPβ with SacI (partial) and XmaI and cloning the FLAG-tagged GABPβ sequence into pTRE-tight (Clontech, Mountain View, CA, USA).

Techniques: Activity Assay, Expressing, Construct, Transfection, Western Blot, Molecular Weight

Journal: Molecular Cancer

Article Title: Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex

doi: 10.1186/1476-4598-10-62

Figure Lengend Snippet: GABPα nuclear localization is rescued by GABPβ in SK-BR-3 cells . MCF-7 and SK-BR-3 cells were transfected with the indicated expression vectors. Cells were stained with anti-FLAG FITC-labeled antibodies (green) and Hoechst dye (blue). Confocal imaging of the overlay of the two stains is shown.

Article Snippet: The final FLAG-GABPβ construct was generated by the ligation of these two fragments. pTRE-tight-GABPβ was prepared by digesting FLAG-GABPβ with SacI (partial) and XmaI and cloning the FLAG-tagged GABPβ sequence into pTRE-tight (Clontech, Mountain View, CA, USA).

Techniques: Transfection, Expressing, Staining, Labeling, Imaging

Journal: Biology

Article Title: Progressive Reduction in Mitochondrial Mass Is Triggered by Alterations in Mitochondrial Biogenesis and Dynamics in Chronic Kidney Disease Induced by 5/6 Nephrectomy

doi: 10.3390/biology10050349

Figure Lengend Snippet: Temporal progression of kidney damage. ( A ) Representative Western blots of renal damage markers and fibrotic markers. ( B ) Quantifications of renal damage markers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), as well as the profibrotic molecule transforming growth factor beta (TGF-β1) and alpha smooth muscle actin (α-SMA). β-Actin was used as loading control. Data are the mean ± SEM, n = 4. Tukey test. a = p ≤ 0.05 vs. sham, b = p ≤ 0.05 vs. 2 days, c = p ≤ 0.05 vs. 4 days, d = p ≤ 0.05 vs. 7 days, e = p ≤ 0.05 vs. 14 days, sham = simulated operation/control group. ( C ) Representative micrographs of kidney: ( C1 ) Normal kidney histology from sham animal. ( C2 ) After 2 days of 5/6 nephrectomy (5/6Nx), there were occasional cortical proximal tubules with flattened epithelium that correspond to tubular atrophy (arrows). ( C3 ) After 1 week of nephrectomy, more tubules showed extensive epithelium damage (arrows). ( C4 ) After 2 weeks of kidney resection, even more tubules show epithelial damage with hyaline casts in their lumen (asterisks). ( C5 ) After 5/6Nx, there were numerous atrophic and dilated tubules (asterisks), as well as a glomerulus with mesangial hypercellularity surrounded by mild inflammatory infiltrate and fibrosis. ( C6 ) The morphometric determination of tubular damage confirmed the progressive tubular damage when compared with the sham simulated/control group (S). (H/E staining, all micrographs 200× magnification, scale magnification bar = 50 µm).

Article Snippet: Anti-guanine adenine (GA)-binding protein subunit beta-1 (GABP-β 1⁄2 or nuclear respiratory factor 2 (NRF2), sc-271571, 1:1000 dilution), anti- nuclear respiratory factor 1 (NRF1) (sc-33771, 1:2000 dilution), anti-neutrophil gelatinase-associated lipocalin (NGAL) (sc-515876, 1:10,000 dilution), anti-profibrotic molecule transforming growth factor beta 1 (TGF-β1) (sc-130348, 1:1000 dilution), anti-dynamin-1-like protein (DRP1) (sc-32898, 1:1000 dilution), anti-mitofusin-1 (MFN1) (sc-50330, 1:1000 dilution), and anti-mitochondrial fission 1 protein (FIS1) (sc-98900, 1:1000 dilution) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Control, Staining

Journal: Biology

Article Title: Progressive Reduction in Mitochondrial Mass Is Triggered by Alterations in Mitochondrial Biogenesis and Dynamics in Chronic Kidney Disease Induced by 5/6 Nephrectomy

doi: 10.3390/biology10050349

Figure Lengend Snippet: Progressive decrease in mitochondrial biogenesis in remnant renal mass. ( A ) Western blots and ( B ) quantifications of mitochondrial proteins: voltage-dependent anion channel (VDAC), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF1) and 2 (NRF2), mitochondrial transcription factor A (TFAM), carnitine palmitoyltransferase 1 (CPT1), and peroxisome proliferator-activated receptor alpha (PPARα). β-Actin was used as loading control. ( C ) Correlation analyses of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), profibrotic molecule transforming growth factor beta 1 (TGF-β1), and alpha smooth muscle actin (α-SMA) with PGC-1α, NRF1, or TFAM. Data are the mean ± SEM, n = 4. Tukey test. a = p ≤ 0.05 vs sham, b = p ≤ 0.05 vs. 2 days, c = p ≤ 0.05 vs. 4 days, d = p ≤ 0.05 vs. 7 days, e = p ≤ 0.05 vs. 14 days, 5/6Nx = 5/6 nephrectomy, d = days after 5/6Nx, sham = simulated operation/control group.

Article Snippet: Anti-guanine adenine (GA)-binding protein subunit beta-1 (GABP-β 1⁄2 or nuclear respiratory factor 2 (NRF2), sc-271571, 1:1000 dilution), anti- nuclear respiratory factor 1 (NRF1) (sc-33771, 1:2000 dilution), anti-neutrophil gelatinase-associated lipocalin (NGAL) (sc-515876, 1:10,000 dilution), anti-profibrotic molecule transforming growth factor beta 1 (TGF-β1) (sc-130348, 1:1000 dilution), anti-dynamin-1-like protein (DRP1) (sc-32898, 1:1000 dilution), anti-mitofusin-1 (MFN1) (sc-50330, 1:1000 dilution), and anti-mitochondrial fission 1 protein (FIS1) (sc-98900, 1:1000 dilution) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Control

Journal: Biology

Article Title: Progressive Reduction in Mitochondrial Mass Is Triggered by Alterations in Mitochondrial Biogenesis and Dynamics in Chronic Kidney Disease Induced by 5/6 Nephrectomy

doi: 10.3390/biology10050349

Figure Lengend Snippet: Evolution of mitochondrial biogenesis and dynamics changes in the time interval between day 2 and day 28 after 5/6 nephrectomy (5/6Nx). In this interval, there is a progressive reduction in the levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor alpha (PPARα), nuclear respiratory factor 1 (NRF1) and 2 (NRF2), and mitochondrial transcription factor A (TFAM), which implies the downregulation of the mitochondrial biogenesis machinery over time. This reduction appears from day 2 and is progressive over time, triggering the decrease in mitochondrial proteins such as voltage-dependent anion channel (VDAC) and carnitine palmitoyltransferase 1 (CPT1) from day 2 to day 28. There is a positive correlation between the reduction in mitochondrial biogenesis factors and the increase in the tubular damage proteins kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) and the fibrotic markers transforming growth factor beta (TGF-β1) and alpha smooth muscle actin (α-SMA). In addition, a slow and gradual change in mitochondrial dynamics from fusion to fission is also shown. This trend started with a reduction on day 2 after 5/6Nx in the fusion proteins mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), favoring the mitochondrial fragmentation observed from 7 days, and continued with an increase in dynamin-1-like protein (DRP1) 28 days after surgery. In addition, macroautophagy markers ubiquitin-binding protein p62 and microtubule-associated proteins 1A/1B light chain 3B I and II (LC3B-I/II) progressively increased with the time, implying the induction of autophagy. This mechanism would principally be present at a chronic stage.

Article Snippet: Anti-guanine adenine (GA)-binding protein subunit beta-1 (GABP-β 1⁄2 or nuclear respiratory factor 2 (NRF2), sc-271571, 1:1000 dilution), anti- nuclear respiratory factor 1 (NRF1) (sc-33771, 1:2000 dilution), anti-neutrophil gelatinase-associated lipocalin (NGAL) (sc-515876, 1:10,000 dilution), anti-profibrotic molecule transforming growth factor beta 1 (TGF-β1) (sc-130348, 1:1000 dilution), anti-dynamin-1-like protein (DRP1) (sc-32898, 1:1000 dilution), anti-mitofusin-1 (MFN1) (sc-50330, 1:1000 dilution), and anti-mitochondrial fission 1 protein (FIS1) (sc-98900, 1:1000 dilution) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Ubiquitin Proteomics, Binding Assay

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Identification of GABP as a Potential Transcriptional Regulator of Diabetic Nephropathy from Proteomic Analysis. A) Proteomic analysis of the differentially expressed proteins in the kidneys of STZ‐induced diabetic mice; B) Gene Ontology analysis of cellular component, biological processes, and molecular function of differently expressed proteins; C) Venn diagram of Protein‐Protein interaction network between differentially expressed proteins and proteins possessing DNA binding transcription factor activity, as defined by the GO term of Mouse Genome Informatics (MGI); D) Expression of GABPα and GABPβ in kidney of two different diabetic mouse models, n = 6; E) Expression level of GABP in major organs of db/m and db/db mice, n = 5. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a two‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to C57 or db/m .

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Binding Assay, Activity Assay, Expressing

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Expression is Specifically Augmented in Mesangial Cells under Diabetic Conditions. A) The protein and B) mRNA expression levels of GABPα and GABPβ in db/m and db/db mice at 8, 16, and 24 weeks of age, n = 5. C) Correlation analysis between GABP expression in serum and UACR in db/m and db/db mice at 8, 16, and 24 weeks of age, n = 6. D,E) Immunohistochemical analysis of GABPα and GABPβ expression in the kidney of db/m and db/db mice at 8, 16, and 24 weeks of age (Scale bar: 50 and 20 µm), n = 6. F,G) Colocalization of GABPα/GABPβ and PDGFRβ by immunofluorescence in glomerular mesangial cells (PDGFRβ, green fluorescence; GABPα/GABPβ, red fluorescence; Scale bar: 20 µm). H) The protein expression levels of GABP in GMCs, endothelial cells (EC), podocytes (PC), and renal tubular epithelial cells (RTEC), n = 3. I) The protein expression levels of GABP in glomerular mesangial cells cultured with different glucose concentrations, n = 3. J) Immunofluorescence was used to detect the protein expression of GABP in glomerular mesangial cells cultured with 0, 10, 20, and 30 m m glucose concentrations (GABPβ, green fluorescence; GABPα, red fluorescence; Scale bar: 100 µm), n = 5. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to db/m or 0 mM glucose concentration.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Expressing, Immunohistochemical staining, Immunofluorescence, Fluorescence, Cell Culture, Concentration Assay

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Overexpression Promotes Mesangial Cell Proliferation and Aggravates Renal Fibrosis of db/m Mice. A) Immunofluorescence and B) its quantitative analysis was used to detect the expression of PCNA, Ki67 in the mesangial cells (PCNA, green fluorescence; Ki67, red fluorescence; Scale bar: 20 µm); n = 3; C) Cell cycle of mesangial cells by flow cytometry. n = 3; D) The protein expression level of Cyclin D, Cyclin E, FN, and COL I in mesangial cells by western blot, n = 3; E) The expressions of LN, FN, COL I and COL IV in mesangial cells by ELISA, n = 5; F) Kidney volume and weight of mice, n = 6; G) BUN, albumin, UACR and α‐MG of mice, n = 6; H) PAS staining (Positive area: red; Scale bar: 20 µm) and transmission electron microscopy (Scale bar: 2 µm) of kidney tissue in mice; I) Sirius red staining (Positive area: red) and Masson staining (Positive area: blue) of kidney tissue in mice, (Scale bar: 20 µm); J) The expression of FN and Collagen I in glomeruli by immunohistochemical staining (Positive area: brown; Scale bar: 20 µm); K,L) The quantitation of Masson and Sirius red postive staining areas, n = 6; M,N) The quantification of Fibronectin and Collagen I expression, n = 6. NG: normal mesangial cell; OV: normal mesangial cell with vector; OE: normal mesangial cell with GABPα/β overexpression lentivirus; db/m : normal control mice; db/m +Veh: db/m mice with vector; db/m +OE: db/m mice with intra‐renal injection of GABPα/β overexpression adeno‐associated virus. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to OV or db/m + V eh.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Over Expression, Immunofluorescence, Expressing, Fluorescence, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Transmission Assay, Electron Microscopy, Immunohistochemical staining, Quantitation Assay, Plasmid Preparation, Control, Injection, Virus

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Knockdown Inhibits Mesangial Cell Proliferation under High Glucose Conditions and Ameliorates Renal Fibrosis of db/db Mice. A) Immunofluorescence and B) its quantitative analysis was used to detect the expression of PCNA, Ki67 in the mesangial cells (PCNA, green fluorescence; Ki67, red fluorescence; Scale bar: 20 µm), n = 3. C) Cell cycle of mesangial cells by flow cytometry. n = 3. D) The protein expression levels of Cyclin D, Cyclin E, FN, and COL I in mesangial cells by western blot, n = 3. E) The expressions of LN, FN, COL I, and COL IV by ELISA, n = 5. F) Kidney volume and weight of mice, n = 6. G) BUN, albumin, UACR, and α‐MG of mice, n = 6. H) PAS staining (Positive area: red Scale bar: 20 µm) and transmission electron microscopy (Scale bar: 2 µm) of kidney tissue in mice. I) Sirius red staining (Positive area: red) and Masson staining (Positive area: blue) of kidney tissue in mice, (Scale bar: 20 µm). J) The expression of FN and Collagen I in glomeruli by immunohistochemical staining (Positive area: brown; Scale bar: 20 µm). K,L) The quantitation of Masson and Sirius red postive staining areas, n = 6. M,N) The quantification of Fibronectin and Collagen I expression, n = 6. NG: normal mesangial cell; HG: mesangial cell with 30 mM glucose; HKV: high glucose cultured mesangial cell with vector; HKD: high glucose cultured mesangial cell with GABPβ‐knockdown lentivirus; db/m : normal control mice; db/db : diabetic model mice; db/db +veh: db/db mice with intra‐renal injection of vector; db/db +KD: db/db mice with intra‐renal injection of GABPβ‐knockdown adeno‐associated virus. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to NG or db/m , # p < 0.05, ## p < 0.01, compared to HKV or db/db + veh.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Knockdown, Immunofluorescence, Expressing, Fluorescence, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Transmission Assay, Electron Microscopy, Immunohistochemical staining, Quantitation Assay, Cell Culture, Plasmid Preparation, Control, Injection, Virus

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: RNA‐seq Identified GLI1 as the Target Gene for GABP. A) Cluster heat map analysis for transcriptomic analysis; B,C) Volcanic map for transcriptomic analysis; D) Classical pathway analysis of GABP overexpress; E) Classical pathway analysis of GABP knockdown; F) Differential gene Venn diagram; G) Differential gene IPA network diagram; H) The nine differential genes generate Gene‐phenotype connections are mapped by VarElect; I) Correlation analysis between GABPα and GLI1; J,K) The mRNA expression levels of GLI1 in the kidney of mice, n = 5; L,M) The protein expression level of GLI1 in the kidney of mice by western blot, n = 5; N,O) Quantitative analysis of Figure L and M, n = 5; P) Immunohistochemical staining (Positive area: brown; Scale bar: 20 µm) and Q,R) its quantitative analysis of GLI1 in the kidney of mice, n = 5. db/m : normal control mice; db/db : diabetic model mice; db/db +veh: db/db mice with intra‐renal injection of vector; db/db +KD: db/db mice with intra‐renal injection of GABPβ‐knockdown adeno‐associated virus. Data are expressed as mean ± s.e.m. Statistical significance was assessed using an unpaired t‐test. * p < 0.05, ** p < 0.01, compared to db/m+ veh, # p < 0.05, ## p < 0.01, compared to db/db +veh.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: RNA Sequencing, Knockdown, Expressing, Western Blot, Immunohistochemical staining, Staining, Control, Injection, Plasmid Preparation, Virus

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Specific Expression of GLI1 in Mesangial Cells and Positive Regulation of GLI1 Expression by GABP in vivo. A) The protein expression levels of GLI1 in glomerular mesangial cells (MC), endothelial cells (EC), podocytes (PC), renal tubular epithelial cells (RTEC), and mononuclear macrophages cells (RAW) n = 3; B) Immunofluorescence staining was used to detect the expression of GLI1 and PDGFRβ in the kidney of db/m and db/db mice (PDGFRβ, green fluorescence; GLI1, red fluorescence; Scale bar: 20 µm); C) The protein and mRNA expression levels of GLI1 in glomerular mesangial cells; D,E) The mRNA expression of GLI1 in mesangial cells; F,G) The protein expression levels of GLI1 in mesangial cells. n = 3; H) ChIP‐qPCR was used to detect the binding of GABP to the GLI1 promoter in mesangial cells; I) Dual luciferase reporter assay of transcription activation for GABPα and β on GLI1; J) Prediction of potential binding sites of GABP and GLI1 via JASPAR; K) Dual‐luciferase reporter assay to detect the predicted binding site of mutant GLI1 to GABP; L,M) The mRNA expression levels of PTCH1, VEGFC, VEGFD and Snail in mesangial cells. NG: normal mesangial cell; OV: normal mesangial cell with vector; OE: normal mesangial cell with GABPα/β‐overexpression lentivirus; HG: mesangial cell with 30 mM glucose; HKV: high glucose cultured mesangial cell with vector; HKD: high glucose cultured mesangial cell with GABPβ‐knockdown lentivirus. Data are expressed as mean ± s.e.m. Statistical significance was assessed using an unpaired t‐test (C, L, M, K) or one‐way ANOVA with Tukey's test (D, E, F, G, I, N) or two‐way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, compared to NG, OV or HKV.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Expressing, In Vivo, Immunofluorescence, Staining, Fluorescence, ChIP-qPCR, Binding Assay, Luciferase, Reporter Assay, Activation Assay, Mutagenesis, Plasmid Preparation, Over Expression, Cell Culture, Knockdown

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Regulates Cell Proliferation and ECM by Inducing GLI1 Expression in Mesangial Cells. A–C) The mRNA expression levels of Collagen I, Fibronectin, and Collagen IV, n = 3; D) The protein expression levels of Collagen I and Fibronectin, n = 3; E,F) The accumulation of Fibronectin and Collagen I in cells was detected by immunofluorescence (DAPI, blue fluorescence; Fibronectin/Collagen I, red fluorescence; Scale bar: 20 µm). NG: normal mesangial cell group (5.56 mmol L −1 glucose concentration); HG: mesangial cell with 30 mM glucose group; HG+DMSO: high glucose cultured mesangial cell with DMSO control group; HG+GANT61: high glucose cultured mesangial cells with 10 µM GANT61 group; GABP: normal mesangial cells with GABPα/β‐overexpression lentivirus; GABP+DMSO: normal mesangial cells with GABPα/β‐overexpression lentivirus and DMSO group; GABP+GANT61: normal mesangial cells with GABPα/β‐overexpression lentivirus and 10 µM GANT61 group; Data are expressed as mean ± s.e.m. Statistical significance was assessed using one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to the NG group, # p <0.05, ## p <0.01, compared to the HG group, @ p < 0.05, @@ p < 0.01, compared to the GABP group.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Expressing, Immunofluorescence, Fluorescence, Concentration Assay, Cell Culture, Control, Over Expression

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Inhibition of GLI1 Effectively Ameliorated GABP‐Mediated Renal Fibrosis in Diabetic Mice. A) Experimental procedure of GANT61 administration in db/m +OE mice; B) Kidney volume and weight of mice, n = 6; C) BUN, Cre, UACR, Cys‐C, α1‐MG and albumin of mice, n = 6; D) Transmission electron microscopy (Scale bar: 2 µm), PAS staining (Positive area: red), PASM staining (Positive area: black), Masson staining (Positive area: blue) and Sirius red staining (Positive area: red) of kidney tissue in mice (Scale bar: 20 µm); E,F) The quantitation of Masson and Sirius red postive staining areas, n = 6; G) The protein expression level of FN and Collagen I in mice kidney by western blot, n = 6; H) Experimental procedure of GANT61 administration in db/db mice; I) Kidney volume and weight of mice, n = 6; J) BUN, Cre, UACR, Cys‐C, α‐MG and albumin of mice, n = 6; K) Transmission electron microscopy (Scale bar: 2 µm), PAS staining (Positive area: red), PASM staining (Positive area: black), Masson staining (Positive area: blue) and Sirius red staining (Positive area: red) of kidney tissue in mice (Scale bar: 20 µm); L,M) The quantitation of Masson and Sirius red postive staining areas, n = 6; N) The protein expression level of FN and Collagen I in mice kidney by western blot, n = 6. db/m : normal control mice; db/m +veh: db/m mice with intra‐renal injection of vector; db/m +OE: d b/m mice with intra‐renal injection of GABPα/β overexpression adeno‐associated vins; OE+GANT61: db/m +OE mice with GANT61 (30 mg kg −1 , once every two days for 3 weeks) administration; db/db : diabetic model mice; db/db +Veh: db/db mice with equivalent dose of the corn oil; db/db +GANT61: db/db mice with GANT61 (30 mg kg −1 , once every two days for 3 weeks) administration. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a one‐way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, compared to OE+GANT61 or db/db +veh, # p < 0.05, ## p < 0.01, compared to db/m or db/db .

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Inhibition, Transmission Assay, Electron Microscopy, Staining, Quantitation Assay, Expressing, Western Blot, Control, Injection, Plasmid Preparation, Over Expression

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Machine Learning Approach Evaluates the Clinical Diagnostic Efficacy of GABP in Diabetic Nephropathy. A) The expression levels of GABP in human serum on healthy control volunteers (HC), type 2 diabetes without any kidney disease (DM), diabetic nephropathy (DN) and membranous glomerulonephritis (MGN) groups; B) The serum expression levels of GABP in different stage of diabetic nephropathy (A1: UACR < 30 mg g −1 , A2: UACR 30–300 mg g −1 , A3: UACR > 300 mg g −1 ); C) Correlation analysis between GABP expression in serum and estimated glomerular filtration rate (eGFR) of patient; D) Correlation analysis between GLI1 expression and eGFR of patient in the Nephroseq; E) Correlation analysis between GLI1 expression and serum creatinine of patient in the Nephroseq; F) The relative expression levels of GLI1 in normal nephrectomy samples adjacent to tumors (Control), different stage of diabetic nephropathy (Early DN: UACR between 30 and 300 mg g −1 , eGFR >90 mL min −1 /1.73 m 2 ; advanced DN: UACR >300 mg g −1 , eGFR <90 mL min −1 /1.73 m 2 ), Data sourced from GSE142025 in the GEO; G,H) The relationship between the model error rate and the number of decision trees without GABP. The red line represents the error rate of DM prediction, the green line represents the error rate of DN prediction, and the black line represents the error rate of out‐of‐pocket samples. I,J) ROC curve of (G, H) model. K,L) The top 10 important variables of (G, H) model. M) ROC curve of several renal function indexes, n = 120. Data are expressed as mean ± s.e.m. Statistical significance was assessed using one‐way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the HC or Control group, ## p < 0.01, compared to the DM group.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Diagnostic Assay, Expressing, Control, Filtration