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. 2017 Aug 8;7:7518. doi: 10.1038/s41598-017-07932-z

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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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In vitro analysis of the effects of phenamil treatment on high doses BMP2-induced inflammation in RAW264.7 cells. (a) Increased inflammatory genes (IL-1α and IL-6) in BMP2 (500 ng/mL) treatment were suppressed by extra phenamil treatment (20 µM) via the increase of Trib3. The level of gene expression was measured by real-time PCR assay; (b) Elisa assay was utilized to detect inflammatory cytokines expression (IL-1α and IL-6); (c) NF-κB activities was examined by luciferase assay; (d) Western-blot assay confirmed phenamil-mediated inflammatory prohibition by increased Trib3 expression to suppress phosphorylated p65 (NF-κB signaling); (e,f) A co-culture system between RAW264.7 and BMSCs showed that phenamil rescued the high-dose BMP2-mediated inhibitory osteogenesis by ALP staining (e) and semi-quantification (f). Scale bar = 200 µm. Ctr, Control; Phe, phenamil; BMP, BMP2; p-p65, phosphorylated-p65. Data presented as means ± SD (n = 3/group); *p < 0.05, **p < 0.01.Uncropped western blot gels are displayed in Supplementary Figure 6.