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. 2017 Jul 26;7:6630. doi: 10.1038/s41598-017-07014-0

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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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Bone matrix components stimulate the expression of inflammasome constituents. BMM from WT or Nlrp3 ^−/− mice were incubated with 50 ng/ml RANKL in the presence of CMG as a source of M-CSF. Cells pre-treated with RANKL for 2days (D2) were exposed to bone particles (BP, 125 µg/ml) or PBS for 24 hours or 72 hours (h). Cells were harvested on D0 (the first day of RANKL stimulation), D2, D3 or D5 and protein and mRNA expression was assessed. (a) Western blot analysis of NLRP3 expression. Data are representative of at least 3 independent experiments. Results are from the same gels, but the lanes were cut and pasted (see S1f). (b–d) qPCR analysis of mRNA expression. Data are expressed as means ± SD, and are representative of at least 3 independent experiments. (b) ****p < 0.0001 (one-way ANOVA and Sidak’s multiple comparison test); (c) WT: ***p < 0.0005, Nlrp3 ^−/−: ****p < 0.0001; (d) ****p < 0.0001. (e) Western blot analysis of pathway activation. WT BMM were serum-starved overnight and stimulated with 125 µg/ml BP. Cells were harvested at the indicated time points.