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. 2020 Jun 8;10:9211. doi: 10.1038/s41598-020-66135-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Smad2/3 activation plays an important role in differentiation of DFAT cells into mural cells. (A,B) Western blotting analysis for phospho-TGFBR1 and phospho-Smad2 expression in DFAT cells. Protein expression of phospho-TGFBR1, phospho-Smad2 in DFAT cells in the presence of TGF-β1 and a Smad2/3 inhibitor PD169316 (A). Effects of PD169316 in different doses on phospho-Smad2 in DFAT cells (B). (C–G) The changes of pericyte marker expressions in DFAT cells on adding DMSO or PD169316 into the culture medium was examined with TGF-β1. Immunofluorescence staining of GFP labelled DFAT cells with ASMA (C) and NG2 (E) in each of the groups. Scale = 30 μm. The percentage of ASMA-positive (D) and NG2-positive (F) cells. The gene expression of ASMA and NG2 in DFAT cells in the presence of TGF-β1 and PD169316 (G). (H) The changes of pericyte marker expressions in DFAT cells in the presence of a Smad2/3 inhibitor or a TGF-β1-neutralizing antibody, in co-culture with MS1 cells. The percentage of ASMA or NG2-positive cells (H). Values are represented as mean ± SD. *p < 0.05.