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. 2020 Apr 24;11:2012. doi: 10.1038/s41467-020-15743-6

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© The Author(s) 2020

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. 8 — a Representative micro-CT images of paraffin embedded lungs of 3-months doxycycline-induced conditional Nedd4-2^−/− and control mice treated with pirfenidone (pirf) or vehicle alone for one month. Fibrotic areas show patches of increased tissue density (white arrowheads) (control, n = 8; conditional Nedd4-2^−/−, n = 8; conditional Nedd4-2^−/− +pirf, n = 10). b Volumetric analysis of fibrotic regions in the three experimental groups (control, n = 8; conditional Nedd4-2^−/−, n = 8; conditional Nedd4-2^−/− +pirf, n = 10). c, d Effect of pirfenidone treatment on static compliance (control, n = 30; conditional Nedd4-2^−/−, n = 28; conditional Nedd4-2^−/− +pirf, n = 27) (c) and on concentrations of active TGFβ in lung homogenates (control, n = 27; conditional Nedd4-2^−/−, n = 24; conditional Nedd4-2^−/− +pirf, n = 24) (d). e–g Number of neutrophils (control, n = 29; conditional Nedd4-2^−/−, n = 25; conditional Nedd4-2^−/− +pirf, n = 25) (e) and concentration of IL-13 in bronchoalveolar lavage fluid (control, n = 21; conditional Nedd4-2^−/−, n = 24; conditional Nedd4-2^−/− +pirf, n = 22) (f), and concentration of IL-1β in lung homogenates (control, n = 15; conditional Nedd4-2^−/−, n = 15; conditional Nedd4-2^−/− +pirf, n = 16) (g) of conditional Nedd4-2^−/− and control mice treated with pirfenidone or vehicle alone. *P < 0.05, **P < 0.01, ***P < 0.001. h–k Effect of pirfenidone on TGFβ signaling in alveolar type 2 cells isolated from conditional Nedd4-2^−/− and control mice induced with doxycycline for 2 weeks that were pretreated with 1 ng/ml TGFβ and analyzed for phosphorylated Smad2 (pSmad2) and transcript levels of TGFβ-regulated genes. h Densitometric analysis of pSmad2 at baseline and after 1–3 h of TGFβ stimulation. i–k mRNA levels of Serpine1 (i), Smad7 (j), and Skil (k) at indicated times after TGFβ stimulation in the presence or absence of pirfenidone. (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 compared to control mice. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to pirfenidone treated conditional Nedd4-2^−/− mice. Statistical analysis was performed with Kruskal wallis test and Mann–Whitney test corrected for multiple comparisons with Bonferroni method as post hoc test in b, d–g, one-way ANOVA with Tukey’s post hoc test in c and two-way ANOVA with Tukey’s post hoc test in h–k. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.