specific inhibitor smad3 (sis3  (Millipore)

Journal: Stem cell research

Article Title: SIPA1 enhances SMAD2/3 expression to maintain stem cell features in breast cancer cells.

doi: 10.1016/j.scr.2020.102099

Figure Lengend Snippet: Fig. 6. SIS3 treatment increases the sensitivity to chemotherapy in breast cancer cells expressing a high level of SIPA1. (A) IC50 for epirubicin and doxorubicin in breast cancer cells treated with or without SIS3. Cells in the presence of either DMSO or SIS3 were treated with epirubicin or doxorubicin for 24 h and cell viability was determined. IC50 for epirubicin and doxorubicin for each cell sample was calculated using GraphPad 8.0. Error bar represents the 95% confidence interval of IC50 (n = 6). (B) A schematic diagram for determining the effect of SIS3 on the growth and chemosusceptibility of MDA-MB-231 tumorspheres. (C) Representative images of MDA-MB-231 tumorspheres after treatment with SIS3 and/or epirubicin in fibrinogen soft gels for up to 10 days. Scale bar, 100 μm. (D) The size of MDA- MB-231 spheroids treated with SIS3 and/or epirubicin in fibrinogen soft gels on days 5, 7 and 10. Mean ± SD, n = 5. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance by unpaired Student’s t-test.

Article Snippet: Breast cancer cells were cultured in RPMI-1640 medium containing 5 μM of epirubicin (Cayman Chemical, Ann Arbor, MI) or doxorubicin (Cayman Chemical), in the presence or absence of 3 μM of the SMAD3-specific inhibitor SIS3 (MedChemExpress, Monmouth Junction, NJ).

Techniques: Expressing

Journal: PLoS ONE

Article Title: High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells

doi: 10.1371/journal.pone.0020319

Figure Lengend Snippet: A: SMAD7 and SERPINE1 promoters were amplified using the ligation-mediated PCR amplified immunoprecipitation product and gene specific primers. (1) Mock IP (anti-flag Ab); (2) anti-SMAD3 Ab (Upstate Biosciences); (3) anti-SMAD2,3 Ab (BD Biosciences). B-G: Enhanced SMAD3 binding to target promoters through exogenous TGFβ1 stimulation. The left panel illustrates baseline promoter binding of SMAD3 and the right panel shows promoter binding after 30 min 2 ng/mL TGFβ1 stimulation. The known SMAD3 target genes SERPINE1 , COL7A1 , SMAD6 , SMAD7 , TGFB1 , and LTBP3 are shown in B-G, respectively.

Article Snippet: Specific Inhibitor of SMAD3 (SIS3, EMD Chemicals, Inc., San Diego, CA) is a potent, specific inhibitor of TGFβ1/ALK-5 phosphorylation of SMAD3 while having no effect on SMAD2, p38 MAPK, ERK, or PI 3-K signaling .

Techniques: Amplification, Ligation, Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells

doi: 10.1371/journal.pone.0020319

Figure Lengend Snippet: Heat map of average expression values for genes known to be affected by the TGFβ1/SMAD3 pathway by microarray analysis. Color intensity values correspond to log 2 of absolute intensity and reach saturation on the heat map at value 4 to preserve dynamic range at lower values. The time series is in hour after TGFβ1 stimulation and vehicle only (DMSO; left) and with TGFβ1 stimulation and also inhibition of SMAD3/ALK5 phosphorylation by Specific Inhibitor of SMAD3 (SIS3) (right). The gene expression profiles on the left (non-SIS3-treated) were all identified as significantly up- or down-regulated (p<0.00001) by STEM as described in the method section. A * indicated microarray results from two distinct DNA probes of the same gene.

Article Snippet: Specific Inhibitor of SMAD3 (SIS3, EMD Chemicals, Inc., San Diego, CA) is a potent, specific inhibitor of TGFβ1/ALK-5 phosphorylation of SMAD3 while having no effect on SMAD2, p38 MAPK, ERK, or PI 3-K signaling .

Techniques: Expressing, Microarray, Inhibition

Journal: PLoS ONE

Article Title: High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells

doi: 10.1371/journal.pone.0020319

Figure Lengend Snippet: A: ChIP SMAD3-bound target genes grouped by signaling pathway and ranked in order of statistical significance. The ratio of genes (orange line) refers to number of genes involved in pathway divided by total genes; approximately 10% of bound genes are identified as belonging to the known TGFβ1 signaling pathway. Other prominent signaling pathways include ERK/MAPK and integrin signaling, which is consistent with known interactions of TGFβ1. Data and image generated using Ingenuity Pathways Analysis. B: Combined gene expression microarray and ChIP-on-chip data grouped by biological process using MetaCore GeneGo systems biology analysis tools , . The top 10 identified pathways are shown. The solid blue and orange bars represent the -log (p -value) for unique genes identified by the TGFβ1-induced gene expression and the ChIP SMAD3-bound genes, respectively. The stripped blue bars represent the -log ( p -value) for common genes identified by both TGFβ1-induced gene expression and ChIP SMAD3-bound genes. The black bars represent the -log ( p -value) for similar genes identified by both TGFβ1-induced gene expression and ChIP SMAD3-bound genes.

Article Snippet: Specific Inhibitor of SMAD3 (SIS3, EMD Chemicals, Inc., San Diego, CA) is a potent, specific inhibitor of TGFβ1/ALK-5 phosphorylation of SMAD3 while having no effect on SMAD2, p38 MAPK, ERK, or PI 3-K signaling .

Techniques: Generated, Expressing, Microarray

Journal: PLoS ONE

Article Title: High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells

doi: 10.1371/journal.pone.0020319

Figure Lengend Snippet: A and B: Quantitative real-time PCR of SERPINE1 (A) and FOXA2 (B) gene expression levels in human A549 cells after 2, 12, and 24 h of stimulation with 2 ng/ml exogenous TGFβ1 and the specific SMAD3 inhibitor, SIS3, or a vehicle-only control (DMSO). The asterisk denotes a highly statistically significant ( p <0.001; n = 3) difference at each time point between SIS3-treated and vehicle-only controls after TGFβ1 treatment. C: Quantitative real-time PCR of SERPINE1 and FOXA2 levels in human Small Airway Epithelial Cells (SAEC) at 2, 12, and 24 h TGFβ1 treatment in relation to control (no TGFβ1). The asterisk denotes a statistically significant ( p <0.01; n = 3) difference at each time point for SERPINE1 and for FOXA2 at 24 h with respect to no TGFβ1 treatment (time zero).

Article Snippet: Specific Inhibitor of SMAD3 (SIS3, EMD Chemicals, Inc., San Diego, CA) is a potent, specific inhibitor of TGFβ1/ALK-5 phosphorylation of SMAD3 while having no effect on SMAD2, p38 MAPK, ERK, or PI 3-K signaling .

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells

doi: 10.1371/journal.pone.0020319

Figure Lengend Snippet: A: ChIP promoter binding profile of FOXA2 , baseline (left) and after 30 min 2 ng/ml TGFβ1 stimulation (right). Each bar height indicates respective array signal intensity for that probe. Values from the three promoter array replicates are shown (green, blue, purple, respectively). If the binding was statistically significant, the binding curve (red) is also included and shows the fitted peak shape. B: Heat map illustration specifically of FOXA2 ChIP binding values (left) with respective gene expression microarray intensities with and without SIS3 treatment (right and far right, respectively). The microarray expression values are plotted in a bar graph (bottom) and show significant repression (white bars) of FOXA2 during a time course of TGFβ1 treatment that is largely abolished by SIS3 treatment (black bars). C: Electrophoretic mobility shift assay shows specific binding of the SMAD3 protein (lanes 2-4) and nuclear extract from TGFβ1-stimulated A549 cells (lanes 5-7). Lanes 3/6 and 4/7 contain non-labeled competitor FOXA2 promoter sequence DNA, 40 ng and 200 ng, respectively. Lane 8 contains a polyclonal Ab against SMAD3 and has a supershift band (3).

Article Snippet: Specific Inhibitor of SMAD3 (SIS3, EMD Chemicals, Inc., San Diego, CA) is a potent, specific inhibitor of TGFβ1/ALK-5 phosphorylation of SMAD3 while having no effect on SMAD2, p38 MAPK, ERK, or PI 3-K signaling .

Techniques: Binding Assay, Expressing, Microarray, Electrophoretic Mobility Shift Assay, Labeling, Sequencing

Journal: Biochemical pharmacology

Article Title: TGF-β1-induced miR-424 promotes pulmonary myofibroblast differentiation by targeting Slit2 protein expression

doi: 10.1016/j.bcp.2020.114172

Figure Lengend Snippet: Interdependence of miR-424 and Smad3 in TGF-β1 profibrogenic signaling. HLFs were treated without (control) or with SIS3 (10 μM) for 1 h prior to treatment with TGF-β1 (1 ng/mL) for 48 h. The cells were harvested for western blot analysis of α-SMA protein (A) and qRT-PCR analysis of miR-424 expression (B). (C) HLFs were transfected with 50 nM of anti-miR-424 or its control for 24 h and then stimulated without or with 1 ng/mL TGF-β1 for 30 min. Cells were harvested for western blot analysis (top) and quantification (bottom) of phosphorylated Smad3 protein (P-Smad3), normalized by total Smad3 in HLFs. Data shown are means ± SEM from three separate experiments, *P < 0.05.

Article Snippet: The Smad3 specific inhibitor SIS3 was purchased from Tocris BioScience (Minneapolis, MN, United States).

Techniques: Control, Western Blot, Quantitative RT-PCR, Expressing, Transfection

Journal: Biochemical pharmacology

Article Title: TGF-β1-induced miR-424 promotes pulmonary myofibroblast differentiation by targeting Slit2 protein expression

doi: 10.1016/j.bcp.2020.114172

Figure Lengend Snippet: Slit2 inhibits TGF-β1-induced cell differentiation. HLFs were pre-transfected with Slit2 specific siRNA or its scrambled control (50 nM) for 24 h and then exposed to 1 ng/mL of TGF-β1 or vehicle for additional 48 h. The cells were then subjected to qRT-PCR analysis of Slit2 mRNA expression (A) or western blot of α-SMA protein expression (B). GAPDH was used as an internal control. Inset in A: Representative gel images of conventional PCR products of Slit2 (188 bp) and GAPDH (206 bp) mRNAs. (C) Left: Immunofluorescence staining images of α-SMA-positive stress fibers (green) and DAPI showing nuclei (blue) of HLFs. Scale bar: 100 μm. Data are means ± SEM from at least 100 fibroblast cells (*P < 0.05). (D) Western blot analysis (left) and quantification (right) of phosphorylated Smad3 protein (P-Smad3), normalized by total Smad3 in HLFs. Data shown in A, B and D are means ± SEM from at least three separate experiments, *P < 0.05 and **P < 0.01.

Article Snippet: The Smad3 specific inhibitor SIS3 was purchased from Tocris BioScience (Minneapolis, MN, United States).

Techniques: Cell Differentiation, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining