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. 2019 Nov 6;9:16092. doi: 10.1038/s41598-019-52627-2

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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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Silencing autophagy genes inhibits TGFβ-induced fibrosis. Three independent strains of human TM primary cells were transfected with siRNAs targeting Atg5/Atg7 (siAtg5/7), LC3 (siLC3), or Smad2/3 (siSmad2/3). A scrambled siRNA (siNC) was used as control. At 2 d.p.t, cells were treated with either TGFβ1 or TGFβ2 (10 ng/mL) for 48 h. (A) Protein levels of SMA, FN1, Atg5, LC3, and Smad2 evaluated by western blot in whole cell lysates (5 μg). (B) Relative protein levels quantification of the fibrotic markers SMA (B) and FN1 (C) calculated from densitometric analysis of the bands, expressed as percentage of siNC_Ctrl (set to 100%). β-actin was used as loading control. (D) Protein levels of FN1 and Col I in conditioned-culture media (20 μL) analyzed by western-blot. (E,F) Specific bands were quantified by densitometry and normalized with total protein (μg) of corresponding whole cell lysates, expressed as percentage of siNC. ^†Denotes statistical significance when comparing TGFβ treatment versus control, using one-way ANOVA with multiple comparisons; ^‡denotes statistical significance when comparing TGFβ-treated siAtg5/7, siLC3 or siSmad2 versus their respective siNC treated control, using two-way ANOVA with Bonferroni post-test. Specific mean ± SD and p values, as well as densitometric analysis confirming significant knocked down expression of LC3, Atg5, and Smad2 are included in Supplemental Material. RPL: Relative protein levels.