Tgf β inhibitor sb 431542

Manufactured by Selleck Chemicals

Sourced in China, Germany, United States

SB-431542 is a highly selective and potent inhibitor of the transforming growth factor-beta (TGF-β) type I receptor ALK5 (activin receptor-like kinase 5). It acts by blocking the phosphorylation of Smad2 and Smad3, which are key mediators of the TGF-β signaling pathway.

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TGF-β (7 citations)

4 protocols using tgf β inhibitor sb 431542

Transforming Growth Factor-β Signaling

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Recombinant human TGF‐β1 was purchased from R&D System. TGF‐β inhibitor SB‐431542 was purchased from Selleck (Shanghai, China). The antibodies for SDPR (ab103230) and N‐cadherin (ab18203) were purchased from Abcam (Cambridge, MA, USA). The antibodies for pSmad2/3 (9510), Smad2/3 (total; 8685), Cyclin D1 (2926), p21 (2946) and β‐catenin (8480) were purchased from Cell Signaling (Beverly, MA, USA). The antibodies for Vimentin (sc‐373717), E‐cadherin (sc‐7870) and β‐actin (sc‐47778) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Tian Y., Yu Y., Hou L., Chi J., Mao J., Xia L., Wang X., Wang P, & Cao X. (2016). Serum deprivation response inhibits breast cancer progression by blocking transforming growth factor‐β signaling. Cancer Science, 107(3), 274-280.

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Proliferation of Pancreatic Beta Cells

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For 5-ethynyl-2′-deoxyuridine (EdU) labelling the islets were adhered to culture plates as described for the cell viability analysis. The proliferation experiments were performed in RPMI-1640 supplemented with 10% (vol./vol.) FCS. MANF (100 ng/ml), TGF-β inhibitor SB431542 (2 μmol/l; Selleck Chemicals, Munich, Germany) and EdU (100 μmol/l; Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA) were added at time zero; 50% of the media was changed every 48 h. After 96 h, the cells were fixed in 4% (wt/vol.) PFA for 20 min and EdU detected by Click-iT EdU Imaging Kit (Invitrogen) according to the manufacturer’s protocol, followed by C-peptide staining. The number of proliferating C-peptide-positive cells was counted from at least five randomly chosen images per donor or by counting all existing beta cells on the culture dish. Each image contained approximately 100–400 beta cells.

Hakonen E., Chandra V., Fogarty C.L., Yu N.Y., Ustinov J., Katayama S., Galli E., Danilova T., Lindholm P., Vartiainen A., Einarsdottir E., Krjutškov K., Kere J., Saarma M., Lindahl M, & Otonkoski T. (2018). MANF protects human pancreatic beta cells against stress-induced cell death. Diabetologia, 61(10), 2202-2214.

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Differentiation and Isolation of iPSC-Derived Endothelial Cells

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Patient-specific iPSCs were generated using the OSKM CytoTune-iPS 2.0 Sendai Reprogramming Kit viral particle factors (Life Technologies) as described previously (17 (link)). The iPSCs used for this study were at passages 22–25. iPSCs were cultured as described above until reaching 80% confluence. The medium was switched to RPMI-B27 without insulin (Life Technologies) with 6 μM CHIR99021 for 2 days, and then changed to 6 μM CHIR99021 for another 2 days. During the differentiation process, from day 4 to day 12, the medium was changed to EGM2 (Lonza) supplemented with 50 ng/ml VEGF (Peprotech), 20 ng/ml BMP4, and 20 ng/ml FGF2 (Peprotech). By day 12, cells were dissociated using TyrpLE for 5 min and sorted using CD144-conjugated magnetic microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD144-positive cells were seeded on 0.2% gelatin-coated plates and maintained in EGM2 medium supplemented with 10 μM TGFβ inhibitor (SB431542) (Selleckchem). After passage 2, iPSC-ECs were cultured in normal EGM2, without SB431542. The iPSC-ECs used for this study were all at passage 3.

Sayed N., Liu C., Ameen M., Himmati F., Zhang J.Z., Khanamiri S., Moonen J.R., Wnorowski A., Cheng L., Rhee J.W., Gaddam S., Wang K.C., Sallam K., Woo Y.J., Rabinovitch M, & Wu J.C. (2020). Lovastatin improves endothelial dysfunction and cellular crosstalk in LMNA-related dilated cardiomyopathy. Science translational medicine, 12(554), eaax9276.

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siRNA Knockdown Experiments with TGF-β Inhibition

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siRNA experiments utilized the following RNAs: Spock1-s224676; EGR2-s4542; IL31RA-s43773; ATP6VD02-s48333, control-4390843 (Invitrogen, United States). Transfection was performed with Lipofectamine RNAiMAX (13778-150; Invitrogen, United States). For TGF-β1 inhibition, TGF-β inhibitor (SB431542; Selleck chem, United States) was used according to the manufacturer’s instructions (10 µM).

Anand G., Clark-Dinovo C., Perry A.M., Goodwin V.M., St. Raymond E., Sakleshpur S, & Steed A.L. (2024). Aromatic amino acid metabolites alter interferon signaling and influenza pathogenesis. Frontiers in Molecular Biosciences, 10, 1232573.

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