Journal: Kidney international

Article Title: Inhibition of transforming growth factor β1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice.

doi: 10.1016/j.kint.2021.02.035

Figure Lengend Snippet: Figure 4 | Lineage tracing for platelet-derived growth factor receptor b–positive (PDGFR-bD) cells was performed using PDGFR- bCreERT2/D mT/mG mice as controls without transforming growth factor b receptor 2 (TGFb-R2) deletion and in mice with tamoxifen- inducible deletion of TGFb-R2 in PDGFR-bD cells (TGFb-R2ko) with the mT/mG reporter. After tamoxifen treatment, all cells with Cre activation start to express green fluorescent protein (GFP). (Top row) Immunohistochemistry for a-smooth muscle actin (a-SMA) (red; left), GFP (green; middle), and merge (right) for lineage tracing of matrix-producing PDGFR-bþ cells on kidney tissue of a TGFb-R2ko mT/mG mouse after 5-day unilateral ureteral occlusion (UUO). Costaining of a-SMA showed a good overlap with GFP in a majority of myofibroblasts identified by a-SMA. These cells were targeted by the deletion of TGFb-R2. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (blue). Bars ¼ 50 mm. (Bottom row) Immunohistochemistry for the active nuclear transcription-factor phosphorylated SMAD3 (pSMAD3) (red) and GFP (green) on kidney sections of PDGFR-bCreERT2/þ mT/mG mice (left) and TGFb-R2ko mice (right) after 5-day UUO. In control kidneys, a robust nuclear pSMAD3 signal as an indicator for an activated TGFb pathway could be detected in tubules and GFPþ interstitial cells (arrows) after 5-day UUO. In TGFb-R2ko mice, GFPþ interstitial cells showed no activated phosphorylated SMAD3 (pSMAD3) signal (arrows), whereas the tubular pSMAD3 signal was preserved. Bars ¼ 20 mm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Article Snippet: Chicken anti– green fluorescent protein (ab13970, Abcam) and rabbit anti– phosphorylated SMAD3 (#9520S, Cell Signaling Technology) immunohistochemistry was performed analogously.

Techniques: Derivative Assay, Activation Assay, Immunohistochemistry, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mucosal Mast Cell-Specific Gene Expression Is Promoted by Interdependent Action of Notch and TGF-β Signaling.

doi: 10.4049/jimmunol.2100112

Figure Lengend Snippet: FIGURE 7. Notch signaling promotes the nuclear localization of SMAD3 and SMAD4. (A) Expression levels of mRNA for Mcpt1, Mcpt2, and Itgae. Error bars indicate mean ± SD; n = 3. **p < 0.005. (B and C) Confocal imaging of FLAG-tagged N2ICD (green), SMAD3 (red) (B), SMAD4 (red) (C), and DAPI-stained nuclei (blue) in BMMCs. BMMCs were trans- duced with retroviral vector expressing N2ICD or mock vec- tor, and the transduced cells were selected by additional culture with puromycin for 5 d (AC). After the selection, mock vector- transduced BMMCs were cul- tured in the presence or absence of TGF-b for 18 h (B and C). Scale bar, 10 mm. Data are from individual experiments that each represents two independent exp- eriments with similar results.

Article Snippet: The cells were incubated with anti-DYKDDDDK/FLAG tag Ab (1E6; FUJIFILMWako Pure Chemical Corporation) and anti-SMAD3 Ab (C67H9; Cell Signaling Technology) or anti-SMAD4 Ab (D3R4N; Cell Signaling Technology) overnight at 4 C, and then incubated with Alexa Fluor 488 conjugated anti-mouse Ab and Alexa Fluor 594 conjugated anti-rabbit Ab for 1 h at room temperature.

Techniques: Expressing, Imaging, Staining, Retroviral, Plasmid Preparation, Selection

Journal: Cancer cell international

Article Title: DUSP5 regulated by YTHDF1-mediated m6A modification promotes epithelial-mesenchymal transition and EGFR-TKI resistance via the TGF-β/Smad signaling pathway in lung adenocarcinoma.

doi: 10.1186/s12935-024-03382-6

Figure Lengend Snippet: Fig. 3 DUAP5 depletion inhibited EMT and TGF-β signaling pathways. (A) Hallmark analysis of DUSP5-related genes was performed by GSEA. (B) The correlation analysis between metastasis and DUSP5, and EMT and DUSP5 in the single-cell sequencing datasets (EXP0066 and EXP0067) of LUAD was based on the CancerSEA website. (C) The pie was used to represent the strength of genes (CDH1, CDH2, DUSP5, SNAIL, TWIST1 and VIM) of LUAD using GSCALite, red: activity; green: inhibition; grey: non-significant. (D) The RNA levels of DUSP5 expression in LUAD cells. (E)The protein levels of DUSP5 expres sion in LUAD cells were examined by Western blotting. (F) The levels of vimentin, N-cadherin and E-cadherin expression were assessed in UT, siNC, si1 and si2 cell lines. (G) The levels of P-smad2, Smad2, P-smad3, Smad3 and snail expression were assessed in UT, siNC, si1 and si2 cell lines. (H) Cell migratory capability was elucidated by a Wound-healing assay. (I) The invasive and migratory abilities were evaluated by Transwell assays. (J) The images display the bioluminescence signal intensities in each BALB/c nude mouse on day 48 after tail vein injection with NC or shDUSP5 cells (left panel). The bar charts present the results of quantitative and statistical analyses of the bioluminescence signal intensities for the NC and shDUSP5 groups (right panel). *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: After that, the primary antibodies DUSP5 (Abcam, ab200708), E-cadherin (Proteintech, 60335-1-lg), N-cadherin (Proteintech, 22018-1-AP), Vimentin (Abcam, ab92547), Snail (Cell Signaling Technology, #3879), Smad2 (Cell Signaling Technology, #5339), Smad3 (Cell Signaling Technology, #9523), P-smad2 (Cell Signaling Technology, #3108), P-smad3 (Cell Signaling Technology, #9520), YTHDF1 (Proteintech, 26787-1-AP) and GAPDH (Proteintech, 60004-1-lg) followed by, a secondary antibody was applied.

Techniques: Protein-Protein interactions, Sequencing, Activity Assay, Inhibition, Expressing, Western Blot, Wound Healing Assay, Injection

Journal: Molecular medicine reports

Article Title: Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

doi: 10.3892/mmr.2014.2912

Figure Lengend Snippet: Figure 1. Analysis of Smad3/4 complex binding to the β3GalT4 promoter. (A) Smad4 binding site (5'‑GTCTAGAC‑3') located between positions ‑788 and ‑795, relative to the transcriptional start point of the β3GalT4 promoter. (B) EMSA. Each lane contained 0.3 nM labeled probe. Smad4 protein (900 ng) was loaded. Smad3 (400, 600, 800 and 1000 ng) was loaded on lanes 3‑6, respectively. (B‑D) Black arrow indicates retarded DNA fragments; white arrow indicates free probes. (C) EMSA to eliminate the nonspecific binding of the Smad3/4 proteins. The labeled probe and 100 or 200‑fold excess of the unlabeled probe were used in competitive assays. A fragment of actin promoter was used as a nonspecific probe. (D) Determination of the binding sites of Smad3/4. Top: Nucleotide sequence of part of the β3GalT4 promoter region and SBE. All probes used were 40 bp. NotI sites were generated at the SBE site to produce a mutated probe. Underlined nucleotides were changed: Bottom: EMSA using the unlabeled probe and mutated or intact DNA fragment. (E) ChIP assays. Input DNA, a DNA fragment immunoprecipitated with anti‑Smad4 antibody (lane ‘S’) and a DNA fragment immunoprecipitated with IgG (lanes ‘‑’) were used as templates for the polymerase chain reaction. A nonspecific DNA region was used as a negative control. Anti‑Pol II antibodies that immunoprecipitated the Pol II‑actin promoter complexes were used as a positive control. β3GalT4, β1,3-galactosyltransferase‑4; EMSA, electrophoretic mobility shift assay; SBE, Smad4 binding element.

Article Snippet: The primary antibodies used were mouse anti-E-cad IgG2a monoclonal antibody and mouse-anti-β catenin IgG1 monoclonal antibody (BD Biosciences, San Jose, CA, USA), mouse anti-N-cad IgG1 and rabbit anti-RNA polymerase II (Pol II) IgG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-vimentin IgG1 monoclonal antibody and anti-β tubulin IgG1 monoclonal antibody (Sigma-Aldrich), rabbit anti-Smad3 mAb IgG monoclonal antibody and anti-Smad4 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA).

Techniques: Binding Assay, Labeling, Sequencing, Generated, Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Positive Control, Electrophoretic Mobility Shift Assay

Journal: Molecular medicine reports

Article Title: Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

doi: 10.3892/mmr.2014.2912

Figure Lengend Snippet: Figure 2. Effect of Smad3 or Smad4 overexpression in NMuMG cells. (A) Morphological changes. Cells were cultured in 6‑well plates and treated with 2 ng/ ml TGFβ for 48 h. Images were captured under phase‑contrast microscopy. (B and C) Expression of EMT markers in the transfected cells. Cells were cultured in 6‑well plates and treated with or without 2 ng/ml TGFβ for 48 h. The cells were harvested and lysed in radioimmunoprecipitation assay buffer. Lysates (10 µg protein/well) were subjected to SDS‑PAGE and western blot analysis and the expression of the markers N‑cad, vimentin, E‑cad and β‑catenin was analyzed by western blot analysis, as described in Materials and methods. Representative western blot analysis results and expression levels relative to Tubulin are expressed as the mean ± standard deviation from triplicate experiments. *0.01

Article Snippet: The primary antibodies used were mouse anti-E-cad IgG2a monoclonal antibody and mouse-anti-β catenin IgG1 monoclonal antibody (BD Biosciences, San Jose, CA, USA), mouse anti-N-cad IgG1 and rabbit anti-RNA polymerase II (Pol II) IgG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-vimentin IgG1 monoclonal antibody and anti-β tubulin IgG1 monoclonal antibody (Sigma-Aldrich), rabbit anti-Smad3 mAb IgG monoclonal antibody and anti-Smad4 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA).

Techniques: Over Expression, Cell Culture, Microscopy, Expressing, Transfection, Radio Immunoprecipitation, Western Blot, Standard Deviation