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: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Figure 2. GABP Expression is Specifically Augmented in Mesangial Cells under Diabetic Conditions. A) The protein and B) mRNA expression levels of GABP𝛼and GABP𝛽in 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 GABP𝛼and GABP𝛽expression 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/GABP𝛽and PDGFR𝛽by

Article Snippet: Antibodies: 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 Adv.

Techniques: Expressing, Immunohistochemical staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Figure 5. RNA-seq Identified GLI1 as the Target Gene for GABP. A) Cluster heat map analysis for transcriptomic analysis; B,C) Volcanic map for transcrip- tomic 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 GABP𝛼and 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

Article Snippet: Antibodies: 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 Adv.

Techniques: RNA Sequencing, Knockdown, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Figure 6. 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 PDGFR𝛽in 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 GABP𝛼and 𝛽on 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: Antibodies: 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 Adv.

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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Figure 7. 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: Antibodies: 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 Adv.

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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Figure 8. Inhibition of GLI1 Effectively Ameliorated GABP-Mediated Renal Fibrosis in Diabetic Mice. A) Experimental procedure of GANT61 administra- tion 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;

Article Snippet: Antibodies: 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 Adv.

Techniques: Inhibition, Transmission Assay, Electron Microscopy, Staining, Quantitation Assay, Expressing, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202407462

Figure Lengend Snippet: Figure 9. 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 m2; advanced DN: UACR >300 mg g−1, eGFR <90 mL min−1/1.73 m2), 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: Antibodies: 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 Adv.

Techniques: Diagnostic Assay, Expressing, Control