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. 2018 Aug 17;8:12354. doi: 10.1038/s41598-018-30799-7

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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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HEMF secreted factors modulate BMP signaling in squamous epithelial cells. (a) pSMAD1/5/8 immunofluorescent staining (Cy2, green) was performed on paraffin-embedded, formalin-fixed sections of 3D organotypic ALI culture established with control serum-free myofibroblast media (SFMM), conditioned SFMM (cSFMM), and cSFMM plus the addition of GREM1 nAb. Representative images of three independent experiments is shown. Images were viewed with a confocal microscope. Magnification 40x. (b) Immunoblots for pSMAD1/5/8 and ID1 were performed on epithelium harvested from 3D organotypic ALI culture. Epithelial pSMAD1/5/8 and ID1 increase in the presence of cSFMM. Addition of GREM1 nAb inhibits the increase in pSMAD1/5/8 and ID1. Tubulin was used as the loading control. Representative immunoblots are shown of three individual experiments. Samples from each experiment were derived at the same time and processed in parallel. Relative densitometry to loading control tubulin is shown. *P ≤ 0.001; **P ≤ 0.02; **P ≤ 0.05; NS, non-significant with ANOVA for multiple comparisons.