Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3-NDRG1 Signaling Pathway.

doi: 10.1002/mco2.70148

Figure Lengend Snippet: FIGURE 6 Western blotting of TGFβ2, Smad2/3, pSmad2/3, E-cadherin, and N-cadherin expression in AGS and MGC803 cells with TGFβ2 interference or overexpression. GAPDH served as the internal control (A). Effects of SIS3 concentration on NDRG1 expression in AGS and MGC803 cells (B). Effect of TGFβ /Smad inhibitor Trabedersen on NDRG1 expression in MGC803 cells (C). Effect of siRNA interference on Smad2/3 expression (D). Interference of Smad2, Smad3, and SMAD2/3 expression on NDRG1 protein and RNA level in AGS and MGC803 cells (E, F). PCR-amplified products from ChIP assays of MGC803 cells were detected by Southern blotting. Input acted as a positive control, while IgG served as a negative control (G, H). Statistical significance is indicated as follows: *p < 0.05, ***p < 0.001, ns: not significant.

Article Snippet: TGFβ/Smad inhibitor Trabedersen and Smad3 phosphorylation inhibitor SIS3 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Expressing, Over Expression, Control, Concentration Assay, Amplification, Southern Blot, Positive Control, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD39 Contributes to the Ability of Cell Invasion in Heterogeneity of Colorectal Cancer

doi: 10.1111/jcmm.70486

Figure Lengend Snippet: The negative feedback of CD39 transcription regulated by SMAD3. (A) WB assay and statistical analysis show the activity of TGF‐β1/SMAD3 signalling pathway and the expression of Collagen I/III affected in HCT116 cells by CD39 knockdown or over‐expression. (B) IF assay show the subcellular localisation of phosphorylated SMAD3 in HCT116 cells. Proportion (P) and brightness (B) of cells with positive p‐SMAD3 and SMAD3 staining were compiled. Brightness value was considered as 0 to 5. Ratio = ∑ 1 10 P p − SMAD 3 × B p − SMAD 3 P SMAD 3 × B SMAD 3 ÷ 10 × 100 % . (C) WB assay and statistical analysis verify the phosphorylation of SMAD3 in HCT116 cells treated with SMAD3 agonist (SRI‐001381) and antagonist (SIS3). (D) WB assay and statistical analysis show the expression of CD39 in HCT116 cells by Collagen I or III over‐expression. (E) ChIP‐qPCR assay show the occupancies of SMAD3, SMAD4, TIM28, PAF1, RNA pol II, SETDB1, H3K9me3 and H3K9ac and p300 on promoter of CD39 in HCT116 cells treated with SMAD3 agonist (SRI‐001381) and antagonist (SIS3). (F) CD39 promoter truncation experiments show the binding affinity of SMAD3 on different truncated promoter regions pulled down by streptavidin. (G) Venn diagram show the binding proteins on CD39 promoter by mass spectrum. SMAD3 agonist can efficiently recruit SETDB1, SMAD3, SMAD4, ATF7IP, TRIM28 and PAF1 to form silencing complex compared with SMAD3 antagonist. (H) WB assay show the SMAD3, SMAD4, SETDB1, TRIM28 and PAF1 as the components in the co‐repressor complex at CD39 promoter in streptavidin pull down product of HCT116 cells with SMAD3 agonist (SRI‐001381) treatment compare to SMAD3 antagonist (SIS3).

Article Snippet: TGFβ/Smad agonist SRI‐011381 (MedChemExpress, China) (10 μM 1 h) or TGFβ/Smad antagonist SIS3 (MedChemExpress) (5 μM 1 h) were treated with cells.

Techniques: Activity Assay, Expressing, Knockdown, Over Expression, Staining, Phospho-proteomics, ChIP-qPCR, Binding Assay

Journal: Cell Death & Disease

Article Title: RUNX1 contributes to the mesenchymal subtype of glioblastoma in a TGFβ pathway-dependent manner

doi: 10.1038/s41419-019-2108-x

Figure Lengend Snippet: The TGFβ signaling pathway enhances the protein interaction between RUNX1 and p-SMAD3/ SUV39H1. a Western blotting analysis of SMAD2, p-SMAD2, SMAD3, and p-SMAD3 expression in four human glioma cell lines. Densitometric analysis of p-SMAD2 and p-SMAD3 is shown. n = 3 per group. b–e The interaction between RUNX1 and SUV39H1/SMAD3 was validated by co-IP. U251 cells overexpressing Flag-RUNX1 or Flag-SMAD3 ( b ) or Flag-SUV39H1 ( c ) in a background of TGFβ stimulation. N9 cells overexpressing Flag-RUNX1 or Flag-SMAD3 ( d ) or Flag-SUV39H1 ( e ) in the presence of LY2109761. f , g Subcellular localization of RUNX1, p-SMAD3 ( f ) and SUV39H1 ( g ) in U251 cells under normal or TGFβ protein conditions. Scale bar, 5 μm. A correlation analysis verified the positional overlap. n = 5 per group. (TGFβ #1 and TGFβ #2 indicate treatment of TGFβ protein for 4 h and 8 h. LY #1 and LY #2 indicate treatment of LY2109761 for 24 h and 48 h. **** p < 0.0001).

Article Snippet: Tumor tissues treated with or without LY2109761 (a TGFβ/SMAD inhibitor, Selleck) and cell lines (N9, N33, TBD0220C, U251) were sequenced at the Beijing Genomics Institute (BGI, Beijing, China) (Table ).

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: RUNX1 contributes to the mesenchymal subtype of glioblastoma in a TGFβ pathway-dependent manner

doi: 10.1038/s41419-019-2108-x

Figure Lengend Snippet: The TGFβ signaling pathway promotes the expression of BCL3, MGP, and POSTN via the RUNX1/p-SMAD3 transcriptional protein complex. a , b Real-time PCR results showing the levels of the RUNX1, BCL3, COL3A1, MGP, and POSTN mRNA after treatment with shRUNX1, shRUNX1 plus TGFβ protein, LV-RUNX1 or LV-RUNX1 plus LY2109761 treatment in U251 cells or N9 cells. c Western blots were performed to analyze the corresponding protein expression level. GAPDH was used as a loading control. d A table showing the distance from the transcript start site and the binding sequence for BCL3, COL3A1, MGP, and POSTN (including the RUNX1 binding sequence). e ChIP analysis of the ability of RUNX1 to bind to the BCL3, COL3A1, MGP, or POSTN promoter using antibodies against RUNX1 in 293T cells. f Luciferase activity is shown for the control reporter, the wild-type reporter and the mutant reporter with the control or shRUNX1 in N9 cells. g Luciferase activity is shown for the control reporter, the wild-type reporter and the mutant reporter with the control or LV-RUNX1 in U251 cell line. h Western blots were performed to analyze the protein expression of p-SMAD3, BCL3, MGP, and POSTN with the treatment of control or siSMAD3. i ChIP analysis of the ability of p-SMAD3 to bind to the BCL3 or MGP promoter (or coding region) using antibodies against p-SMAD3 in 293T cells. j , k ChIP-PCR analyses showing the changes that occurred in the ability of RUNX1 to bind the promoters of target genes after U251 or N9 cells were treated with TGFβ or LY2109761, respectively. l , m ChIP-PCR analyses showing the changes that occurred in the ability of p-SMAD3 to bind the promoters of target genes after N9 and U251 cells were treated with LY2109761 or TGFβ, respectively. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively).

Article Snippet: Tumor tissues treated with or without LY2109761 (a TGFβ/SMAD inhibitor, Selleck) and cell lines (N9, N33, TBD0220C, U251) were sequenced at the Beijing Genomics Institute (BGI, Beijing, China) (Table ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Binding Assay, Sequencing, Luciferase, Activity Assay, Mutagenesis

Journal: Cell Death & Disease

Article Title: RUNX1 contributes to the mesenchymal subtype of glioblastoma in a TGFβ pathway-dependent manner

doi: 10.1038/s41419-019-2108-x

Figure Lengend Snippet: The TGFβ signaling reduces the expression of MXI1 via the RUNX1/ SUV39H1 transcriptional complex, and regulates the expression of target genes (BCL3, MGP, and MXI1). a , b Real-time PCR showing the effect of shRUNX1, shRUNX1 plus TGFβ, LV-RUNX1, or LV-RUNX1 plus LY2109761 on MXI1 mRNA levels in U251 or N9 cells. c Western blots were performed to analyze the protein expression of MXI1 and H3K9me3 after treatment with shRUNX1/LV-RUNX1 or shSUV39H1/LV-SUV39H1 or rescue with TGFβ/LY2109761. GAPDH and H3 were used as loading controls. d Western blots were performed to analyze the protein expression of MXI1 and H3K9me3 after treatment with H3K9me3 inhibitor. GAPDH and H3 were used as loading controls. e ChIP-qPCR results showing the level of H3K9me3 on the MXI1 promoter after treatment with shRUNX1 or shRUNX1 plus TGFβ. f The level of H3K9me3 enrichment on the MXI1 promoter after LV-RUNX1 or LV-RUNX1 plus LY2109761. g , h Real-time PCR showing the effect of LY2109761 or TGFβ on BCL3, MGP, POSTN, and MXI1 mRNA levels in U251 or N9 cells. i Western blots showing the effect of LY2109761 or TGFβ on BCL3, MGP and MXI1 protein levels in U251 or N9 cells. (* p < 0.05, *** p < 0.001 and **** p < 0.0001, respectively).

Article Snippet: Tumor tissues treated with or without LY2109761 (a TGFβ/SMAD inhibitor, Selleck) and cell lines (N9, N33, TBD0220C, U251) were sequenced at the Beijing Genomics Institute (BGI, Beijing, China) (Table ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, ChIP-qPCR

Journal: Cell Death & Disease

Article Title: RUNX1 contributes to the mesenchymal subtype of glioblastoma in a TGFβ pathway-dependent manner

doi: 10.1038/s41419-019-2108-x

Figure Lengend Snippet: RUNX1 promotes PDX tumor proliferation and invasion in a TGFβ pathway-dependent manner in vivo. a First, nude mice were intracranially implanted with differently treated PDX tissues (control, shRUNX1, and LV-RUNX1). After 4 days LY2109761 and TGFβ were administered via the tail vein, the nude mice were divided into 5 groups, and bioluminescence imaging was performed. b Representative bioluminescence images of mice implanted with intracranial tumors on days 7, 12, 17 and 22. The relative bioluminescence values are shown ( c ). Survival was also measured in these 5 groups ( d , e ). (*, ** and *** indicate p < 0.05, p < 0.01and p < 0.001, respectively). f IHC was used to analyze the expression of RUNX1 at the control, shRUNX1 and LVRUNX1 sites.Quantitative analyses were performed using ImageJ software for each high-magnification view. n = 5 per group. g IHC staining for BCL3, MGP, MXI1 and MMP9 was performed in mouse brain slices in the control, shRUNX1, TGFβ protein plus shRUNX1, LV-RUNX1 and LY2109761 plus LV-RUNX1 groups. h Quantitative analyses of these genes were performed using ImageJ software for each high-magnification view. n = 5 per group. Scale, 20 μm. (** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively).

Article Snippet: Tumor tissues treated with or without LY2109761 (a TGFβ/SMAD inhibitor, Selleck) and cell lines (N9, N33, TBD0220C, U251) were sequenced at the Beijing Genomics Institute (BGI, Beijing, China) (Table ).

Techniques: In Vivo, Control, Imaging, Expressing, Software, Immunohistochemistry

Journal: International journal of molecular medicine

Article Title: AST IV inhibits H₂O₂-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway.

doi: 10.3892/ijmm.2015.2188

Figure Lengend Snippet: Figure 4. Inhibition of the TGF-β1/Smad2 pathway decreases H2O2-induced human umbilical vein endothelial cell (HUVEC) apoptosis, but not oxidative stress. (A) FACS analysis was performed to determine intercellular reactive oxygen species (ROS) production in the HUVECs in the control, model, astragaloside Ⅳ (AST Ⅳ) and LY2109761 groups. (B) Western blot analysis was performed to determine protein expression in the HUVECs in the control, model, AST Ⅳ and LY2109761 groups. (C) RT-qPCR was performed to determine mRNA expression in the HUVECs in the control, model, AST Ⅳ and LY2109761 groups. (D) Annexin V-FITC/PI staining was performed to determine the apoptotic rate of the HUVECs in the control, model, AST Ⅳ and LY2109761 groups. **P<0.01 vs. control group; ##P<0.01 vs. model group. Nox4, NADPH oxidase 4; TGF-β1, transforming growth factor-β1.

Article Snippet: In addition, some cells were treated with diphenyliodonium (DPI, a specific inhibitor of Nox4; from Sigma, St. Louis, MO, USA) or LY2109761 (a selective inhibitor of TGF-β1/ Smad2; from MedChem Express, LLC, Princeton, NJ, USA).

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

Journal: Cancer Management and Research

Article Title: Aberrant CEACAM19 expression is associated with metastatic phenotype in penile cancer

doi: 10.2147/CMAR.S192385

Figure Lengend Snippet: Notes: ( A ) The effect of SB431542 (SB) on p-Smad2/3 levels in Penl1 cells. Penl1 cells were treated with 10 µM SB for 24 hours. β-Actin served as a loading control. ( B ) The effect of SB431542 on wound healing of Penl1 cells. Three different areas in each assay were chosen to measure the distances of migrating cells to the origin of the wound. * P <0.05, Control vs SB. ( C ) The effect of SB431542 on cell invasion of Penl1 cells. Transwell invasion assay was conducted to evaluate cell migration and invasion capability. * P <0.05, Control vs SB. ( D ) Inhibition on TGF-β/Smad2/3 activity by SB431542 reduced MMP2/9 secretion in Penl1 cells. All experiments were performed three times, and data are presented as mean ± SD. values. * P <0.05, Control vs SB.

Article Snippet: As shown in , p-Smad2/3 level was potently reduced by TGF-β/Smad2/3 pathway inhibitor SB431542 (Cell Signaling).

Techniques: Control, Transwell Invasion Assay, Migration, Inhibition, Activity Assay