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. 2018 Aug 27;8:12920. doi: 10.1038/s41598-018-31332-6

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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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Time-dependent phenotype modifications in ALI MCCs: (A) Time-dependent changes in: ATP levels, percentage (%) of live A549 cells, % of LY passage and P_app values in ALI multilayered mono-cultures grown up to 14 d. Data are shown as average ± standard error of the mean (n_replicates = 2; n_tests = 3). The symbols (**) and (***) indicate statistically significant changes as compared to the values measured at 24 h (p < 0.01 and 0.001, respectively) (two-way ANOVA and Bonferroni post-test). (B) Western blot analysis of E-cadherin (epithelial marker), vimentin and fibronectin (mesenchymal markers) in A549 cells forming ALI multilayered mono-cultures and cultured up to 14 d. The time-points examined were: 24 h, 48 h, 72 h, 7 d and 14 d. Abbreviations “n₁” and “n₂” indicate different experimental replicates. β-actin expression is also reported as proteins loading control.