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. 2019 Dec 20;7:351. doi: 10.3389/fcell.2019.00351

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Copyright © 2019 Gao, Zhang, Dai, Zhang, Li and Bai.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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SB-431542 inhibits beta cell differentiation from MSCs by blocking the TGF-β/Smad pathway. (A,B) Duolink PLA fluorescence assay of the interactions of p-Smad2 or p-Smad3 with Smad4 in MSCs following different treatments. Colocalization of p-Smad2 or p-Smad3 with Smad4 around and within nuclear foci is shown in the upper panel, which corroborated the interaction of these proteins in uninduced and induced MSCs (scale bar = 50 μm). The percentages of PLA-positive cells following the different treatments are shown in the bottom panel. The number of positive cells was significantly elevated after treatment with the cocktail of factors compared with uninduced MSCs or SB-431542-treated induced MSCs. (C) Western blotting showed that SB-431542 down-regulated p-Smad2 and p-Smad3, which led to decreased levels of intranuclear Smad4. (D) Flow cytometry analysis of Pdx1 and Ngn3 expression showed that the percentage of Pdx1- and Ngn3-positive induced MSCs was markedly decreased after SB-431542 treatment. (E) RT-qPCR demonstrated that SB-431542 prevented activation of the TGF-β/Smad pathway leading to reduced expression of miR-375 and miR-26a. (F) Glucose-stimulated insulin secretion (GSIS) of induced MSCs after SB-431542 treatment indicated that insulin secretion under conditions of 20.5 mM glucose increased to a much smaller degree than that in cells not treated with the inhibitor (n = 30; paired two-tailed t-test).