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. 2018 Sep 24;8:14299. doi: 10.1038/s41598-018-32653-2

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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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Characterization of D2.0R dissemination to bone. (a) Representative hematoxylin and eosin (H&E) images of D2.0R-inoculated tibiae from −E2 (n = 8 mice) and +E2 (n = 6 mice) mice. Left panels = 4X, right panels = 20X of same tibiae. Scale bars = 500 μM (left panel) and 100 μM (right panel). (b) Histomorphometric analysis of bone volume/total volume (%BV/TV) from mice described in (a) and non-tumor-inoculated (naïve) mice (n = 10 −E2 mice, n = 8 +E2 mice). (c) Representative microCT images of mice described in (a) and (b). (d) microCT analysis of mice described in (c). (e) qPCR analysis of whole bone homogenates from non-tumor-inoculated mice and D2.0R-inoculated mice described in (a) and (b) for Krt18, normalized to mouse B2m. (f) Positive pan-cytokeratin (AE1/AE3) staining in the tibia from a D2.0R-inoculated mouse (mouse number 5). Arrows indicate cytokeratin-positive tumor cells. Left panel = 4X, right panel = 40X of the same tibia. Scale bars = 500 μM (left panel) and 100 μM (right panel). (b,d,e) One-way ANOVA with Sidak’s multiple comparisons test, *P < 0.05, ***P < 0.001 and ****P < 0.0001.