phosphorylated p smad2 3  (Cell Signaling Technology Inc)

Journal: Investigative Ophthalmology & Visual Science

Article Title: The TCF4 Gene Regulates Apoptosis of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy

doi: 10.1167/iovs.66.3.16

Figure Lengend Snippet: Effect of TCF4 knockout on TGF-β2–mediated ECM production and apoptosis. ( A ) The bHLH region or 20 bases in exon 9 of TCF4 in iFECD were knocked out using CRISPR/Cas9 (iFECD TCF4 −/− , iFECD TCF4 ΔbHLH). Cells were cultured in serum-free medium for 24 hours, then treated with or without TGF-β2 (10 ng/mL) for 24 hours. Phase-contrast images show that iFECD forms a monolayer with polygonal morphology. TGF-β2–induced cell death in iFECD but not in iFECD TCF4 −/− or iFECD TCF4 ΔbHLH. Scale bar : 200 µm. ( B ) Sanger sequencing confirmed the deletion of 20 bases in exon 9 of TCF4 in iFECD TCF4 −/− . Red lines indicate the deleted bases. ( C ) Western blotting showed TGF-β2–induced cleavage of caspase-3 and PARP in iFECD, which was suppressed in iFECD TCF4 −/− and iFECD TCF4 ΔbHLH. ( D ) Flow cytometric analysis of Annexin V–positive apoptotic cells in response to TGF-β2 treatment. TGF-β2 treatment substantially increased the percentage of Annexin V–positive cells to 31.4% ± 2.0% in iFECD cells. Both iFECD TCF4 −/− and iFECD TCF4 ΔbHLH cells demonstrated resistance to TGF-β2–induced apoptosis, showing lower percentages of Annexin V–positive cells (19.8% ± 1.3% and 18.0% ± 1.6%, respectively; P = 5.28 × 10 −2 and P = 3.02 × 10 −2 , compared to TGF-β2–treated iFECD). Data are presented as mean ± SEM from three independent experiments. ( E ) Western blotting confirmed suppression of TCF4 in both iFECD TCF4 −/− and iFECD TCF4 ΔbHLH. TGF-β2 upregulated Snail1 in iFECD, but this upregulation was suppressed in both mutant cell lines. ZEB1 expression was unaffected by TGF-β2 in all cell lines. Fibronectin levels increased in iFECD but not in either iFECD TCF4 −/− or iFECD TCF4 ΔbHLH with TGF-β2 treatment. ( F ) Phosphorylation of Smad2 and Smad3 by TGF-β2 was confirmed in both iFECD and iFECD TCF4 −/− , while this phosphorylation was suppressed in iFECD TCF4 ΔbHLH. All experiments were performed independently at least three times with reproducible results.

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto PVDF membranes, which were then blocked with 3% nonfat dry milk for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies against cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), cleaved poly (ADP-ribose) polymerase (cleaved PARP) (1:1000; Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3000; Medical & Biological Laboratories Co., Ltd., Tokyo, Japan), TCF4 (1:500), Snail1 (1:1000; Cell Signaling Technology), ZEB1 (1:1000; Cell Signaling Technology), fibronectin (1:20,000; BD Biosciences), phosphorylated Smad3 (p-Smad3) (1:1000; Cell Signaling Technology), Smad2 (1:1000; Cell Signaling Technology), phosphorylated Smad2 (p-Smad2) (1:1000; Cell Signaling Technology), and Smad3 (1:1000; Cell Signaling Technology).

Techniques: Knock-Out, CRISPR, Cell Culture, Sequencing, Western Blot, Mutagenesis, Expressing, Phospho-proteomics

Journal: Ecotoxicology and environmental safety

Article Title: A novel method for real-time inhalation toxicity assessment in mice using respirometric system: A promising tool for respiratory toxicology.

doi: 10.1016/j.ecoenv.2024.117333

Figure Lengend Snippet: Fig. 7. Histological changes and modulation of the TGF-β/SMAD/COL1A1 signaling pathway in a bleomycin-Induced IPF model in C57/BL6 mice. (A) The histo logical changes measured by H&E staining in lung tissue of bleomycin-induced IPF mice. Scale bars, 300 μm. (B-F) Expression and ratio of TGF-β/GAPDH, p-SMAD2/ SMAD2, p-SMAD3/SMAD3, COL1A1/GAPDH and Caveolin-1/GAPDH were investigated using western blot analysis. Statistical analysis was performed using one- way ANOVA. *p<0.05, ***p<0.001 versus the BLM.

Article Snippet: Anti-TGF-β, anti-SMAD2, anti-phosphorylated SMAD2 (p-SMAD2), anti-SMAD3, anti-phosphorylated SMAD3 (pSMAD3), anti-Caveolin-1, and anti-GAPDH antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Staining, Expressing, Western Blot