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. 2018 Aug 14;8:12123. doi: 10.1038/s41598-018-30640-1

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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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Tumor formation and metastasis by PyMT-1099 cells. (A) PyMT-1099 cells untreated or treated with TGFβ for >20 days (PyMT-1099 LT) were injected orthotopically into mammary fat pads of NSG mice. The graph represents tumor growth in PyMT-1099 and PyMT-1099 LT group of mice. (B) Histological tumor sections from tumors of PyMT-1099 or PyMT-1099 LT cells described in (A) were stained with H&E to assess the morphology of primary tumors. Representative microphotographs are shown from tumors of 2 out of the 6 mice used in the experiment. (C) Immunofluorescence analysis was performed to assess the expression of the EMT markers FN1, VIM, E-CAD and N-CAD in tumors formed by PyMT-1099 or PyMT-1099 LT cells in the experiment described in (A). DAPI was used as a nuclear counterstain. Representative pictures are shown from tumors of 2 out of the 6 mice used in the experiment. Scale bar, 100 μm. (D) The graph represents the number of lung metastases formed in NSG mice orthotopically transplanted with PyMT-1099 or PyMT-1099 LT cells; n = 6. (E) The graph represents the number of lung metastases formed in NSG mice injected with PyMT-1099 or PyMT-1099 LT cells through the tail vein; n = 6. The mice were sacrificed 8 weeks post-injection, and lungs were resected for the analysis of cancer cell colonization/ metastases formation.