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. 2017 Jul 28;7:6847. doi: 10.1038/s41598-017-07126-7

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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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Stiffness of the ECM affects cellular behaviour and gene expression levels. (a) Phase contrast images of H1299 cells incubated on PAGs with the indicated stiffness or on glass. Scale bars, 50 μm. (b) Immunostaining of F-actin (red) and vinculin (green) in H1299 cells incubated under the indicated conditions (left); quantification of vinculin (n = 50) area for each condition (right). (c) Western blot analysis of pFAK, FAK, pERK, ERK, pMLC, and MLC in H1299 cells incubated under the indicated conditions. (d) Quantification of proliferation of H1299 cells incubated on a soft or stiff PAG with the indicated stiffness or on glass for the indicated periods. (e) Microarray gene expression analysis of H1299 cells incubated under the indicated conditions for 24 h. Heat map and gene ontology (GO) analysis of gene expression profiles of 557 upregulated genes with a ≥1.4× change in expression. (f) Heat map representation of microarray data showing the expression levels of several key EMT-related genes. (g) Quantitative RT-PCR (qRT-PCR) analysis of expression of EMT-related genes in H1299 cells incubated on a PAG with 0.5 or 40 kPa stiffness. qRT-PCR values were normalised to GAPDH. Data in (a), (b), (c), (d) and (g) represent the mean of three independent experiments ± SEM. ^* P < 0.05, ^** P < 0.01.