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. 2020 Jan 29;11(1):72. doi: 10.1038/s41419-020-2279-5

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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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Fig. 3 — a The recombinant adenovirus Ad-RFP (Red), Ad-BMP9 (green), and Ad-Sim-Shn3 (red) were shown to effectively transfect hAMSCs for 24 h (scale bar = 100 μm). Effective knockdown of Shn3 expression. b The Ad-Sim-Shn3 expressing siRNA targeting Shn3 transduces hAMSCs with high efficiency after transfection. Ad-Sim-Shn3 silences the expression of Shn3 from 48 to 120 h. All samples were normalized with the house-keeping gene GAPDH. Each experiment was done in triplicate, Ad-Sim-Shn3 vs. Ad-RFP groups. c, e Silencing Shn3 promotes BMP9-induced ALP activity in hAMSCs. ALP biochemical quantification assay (c) and ALP staining assay (e) were conducted to detect the ALP activity under the treatment as shown at 3 and 7 days after transfection (Scale bar = 100 μm). d, f The quantification of mineralization (d) and calcium deposition is observed by Alizarin Red S staining assay (f) under the treatment as shown at 14 and 21 days after transfection (scale bar = 100 μm). g–k Shn3 inhibits the expression of osteogenic relative factors of hAMSCs. g RT-qPCR assay was performed to determine that Shn3 inhibits the expression of osteogenic relative factors, including Runx2, BSP, COL-1, and OSX, induced by BMP9 of hAMSCs at 7 days. h–k Western blotting assay was adopted to detect that Shn3 inhibits the expression of osteogenic relative factors OCN (h), OPN (i), and Runx2 (j) induced by BMP9 of hAMSCs. β-ACTIN served as the loading control. The quantification results of western blotting assay showed the effect of Shn3 on the expression level of OCN, OPN, and Runx2 on BMP9-induced hAMSCs (k). The data are shown as mean ± SD for triplicate. *P < 0.05.