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. 2019 Oct 23;9:15224. doi: 10.1038/s41598-019-51665-0

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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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PA200 is a negative regulator of myofibroblast differentiation. (a) mRNA expression analysis of PA200 (PSME4), cyclin D1 (CCND1), TGF-β1 (TGFB1) and αSMA (ACTA2) in phLF after 24, 48 and 72 h of PA200 silencing. HPRT served as housekeeping gene and expression was normalized to time-matching controls (one-sample t-test, phLF from n = 3 organ donors). (b) Protein expression analysis of PA200, myofibroblast marker α-smooth muscle actin (αSMA) and proliferation markers cyclin D1 (CCND1) and proliferating cell nuclear antigen (PCNA) in phLF transfected with PA200 or control siRNAs for 72 h. Bar diagram shows densitometric analysis of Western blots with normalization of obtained signals to control siRNA-transfected phLF of the same donor (one sample t-test, phLF from n = 4 different organ donors). (c) Cell count of phLF analyzed 72 h after PA200 silencing and normalized to mean of control siRNA-transfected cells (Mann-Whitney U test, phLF from n = 4 different organ donors with three technical replicates per condition). (d) Analysis of PA200 protein levels in phLF upon PA200 overexpression for 24 h (one-sample t-test, phLF from n = 3 different organ donors). (e) Analysis of cell count in phLF under the same conditions used in (d) (one-sample t-test, phLF from n = 3 different organ donors). (f) mRNA expression analysis of Psme4 (PA200), and myofibroblast markers Acta2 (αSMA) and Col1a1 in wildtype and PA200−/− primary mouse lung fibroblasts. Hprt served as house keeping gene (Mann-Whitney U test, pmLF from n = 4 animals per group). Full-length immunoblots are presented in Supplementary Fig. S5.