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. 2019 Sep 27;10:4404. doi: 10.1038/s41467-019-12108-6

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© The Author(s) 2019

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 — CXCL5 and CXCR2 contribute to the regulation of proliferation. a RT-qPCR analysis for CXCL5 expression of bone marrow of healthy mice and PyMT cancer cells. b In situ hybridization (ISH) analysis of paraffin sections of healthy or IC mouse bone co-cultured for 2 weeks with PyMT cells. ISH fluorescent probe (red) was used to detect RNA transcripts in the bone marrow and in Keratin+ cancer cells (green). DapB ISH probe was used as a negative control (scale bar: 10 μm). c IHC staining for CXCL5 and CXCR2 of healthy and IC bone co-cultures injected with PyMT cells (scale bars: Low magnification = 20 μm; High magnification = 10 μm). d Experimental design of ex vivo bone co-cultures. (Top) Healthy mouse bone explants supplemented daily with rCXCL5 protein. (Middle) Healthy mouse bone explants cultured for 2 weeks to achieve a low-proliferative phenotype of cancer cells. These cultures were subsequently supplemented daily with rCXCL5 protein for the next 2 weeks. (Bottom) Cancer-primed bone explants using bones from animals with intracardiac (IC) injection of PyMT cancer cells prior to culture and supplemented daily with CXCR2 antagonist SB225002. e Percentage of Ki67+Keratin+ cancer cells over total number of Keratin+ cancer cells of mouse bone co-cultures (Left) between healthy bone with normal media and healthy bone with rCXCL5 complemented media (p < 0.0001, Student’s t test), (Middle) between healthy bone with normal media and healthy bone with rCXCL5 complemented media 2 weeks after start of co-culture (p = 0.01, Student’s t test), and (Right) between cancer-primed bone with normal media and cancer-primed bone with a CXCR2 antagonist complemented in the media (p = 0.006, Student’s t test). Each dot represents the percentage of Ki67+Keratin+ cancer cells detected in one section of bone. Lines show the mean and standard deviation. f IHC staining for CXCL5 and CXCR2 of a metastatic human femoral head from a patient with breast cancer metastasis to bone. CXCL5 and CXCR2 were found in the human bone samples (scale bar: 20 μm)