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. 2018 Aug 1;9:3016. doi: 10.1038/s41467-018-05274-6

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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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Fig. 3 — Twist1, Prrx1, and TNC formed a positive feedback loop in fibroblasts. a, b Prrx1 directly upregulates TNC expression in fibroblasts. a Both Prrx1a and Prrx1b increased the TNC protein level in normal fibroblasts (SNF#14 and #32). Prrx1 depletion led to the downregulation of TNC expression in CAFs (SCAF#32 and #39). b Prrx1 enhanced the TNC promoter activity. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.01). c, d Prrx1 is indispensable for Twist1-induced TNC expression at the protein (c) and mRNA (d) level. Data are presented as the mean ± SEM; N = 4 independent experiments (two-tailed t test: ***p<0.0001). e, f To confirm the Twist1-Prrx1-TNC PFL, we introduced a doxycycline-inducible Twist1 expression vector into two fibroblast cell lines (MRC5 and IMR90) as well as a normal fibroblast line (SNF#19). Induced Twist1 expression using this Tet-ON system resulted in sequential increases of the Prrx1 and TNC protein (e) and mRNA (f) expression in normal lung fibroblasts (MRC5). g Schematic diagram of the Twist1-Prrx1-TNC PFL