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. 2018 Oct 17;6:30. doi: 10.1038/s41413-018-0031-x

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© The Author(s) 2018

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. 6 — Upregulated CD200 may contribute to decreased osteoclastogenic activity in Gli1-Cre^ERT2;Bmpr1a^fl/fl sutures. a FACS analysis of CD200 in suture mesenchymal cells collected from Bmpr1α^fl/fl (control) and Gli1-Cre^ERT2;Bmpr1α^fl/fl (mutant) mice 2 weeks post induction. b Quantitation of the percentages of CD200+ cells in suture mesenchyme from three independent samples. c Immunostaining of CD200 (green, indicated by arrows) in the suture mesenchyme of Bmpr1α^fl/fl (control) and Gli1-Cre^ERT2;Bmpr1α^fl/fl (mutant) mice 2 weeks post induction (2wpt). Broken lines indicate the outline of the suture. d TRAP staining of osteoclasts induced from BMMs of 4-week-old C57BL/6J mice, after culture with M-CSF for 3 days and then with RANKL or RANKL plus exogenous CD200 for another 5 days. The inset shows a magnified view of the boxed region. Blue arrows indicate mature osteoclasts. e Quantitation of multinucleated TRAP+ osteoclasts per well in four independent experiments. f Resorption activity assay (von Kossa staining) of osteoclasts induced from BMMs of 4-week-old C57BL/6J mice, after culture with M-CSF for 3 days and then with RANKL or RANKL plus exogenous CD200 for another 5 days. g Quantitation of the percentage of resorption in four independent experiments. h Real-time PCR of hedgehog signalling members (Ihh, Ptch1, and Gli1), osteogenic markers (Alp, Runx2, and Sp7), and osteoclastic-related markers (CD200, CD200R, OPG, and RANKL) in the suture mesenchyme of C57BL/6J mice 2 weeks post treatment with or without GDC0449 (hedgehog inhibitor) from five independent samples. T tests were performed. *P < 0.05; **P < 0.01. Scale bars, 100 µm