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. 2017 Jul 25;7:6373. doi: 10.1038/s41598-017-06548-7

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© The Author(s) 2017

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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Activation of Smad1/5/8 signaling pathway is involved in BMP9-induced osteogenic differentiation of rDFCs. (A) Western Blot assay showed that BMP9 activated the Smad1/5/8 signaling pathway and increased the expression of phosphorylation Smad1/5/8. (B) ALP activity assay showed effect of Smad1/5/8 signaling pathway inhibition on rDFCs osteogenic differentiation by Compound C in a dose-dependent manner. (C) Effect of Smad1/5/8 signaling pathway inhibition on osteogenic differentiation of rDFCs in a concentration manner. ALP staining on day 7 was showed. Black bars: 100 μm. (D) Alizarin red staining was used to detect the calcium deposition on days 14 and 21. Black bars: 100 μm. (E,F,G and H) osteogenic markers detected by Real Time-qPCR on days 3, 5, 7, 9. (E) Alkaline phosphatase, ALP. (F) Osterix, (G) Osteopontin, OPN. (H) runt-related transcription factor 2, Runx2. *P < 0.05, compared with blank group, **P < 0.01, ***P < 0.001 compared with blank group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with BMP9 group.