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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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Smad2/3 mediates the dysregulation of autophagy by TGFβ in TM cells. Three independent strains of human TM primary cells were transfected for 72 h with 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 LC3 and Smad2 were evaluated by western blot in whole cell lysates (5 μg). (B,C) Relative protein levels quantification were calculated from densitometric analysis of the bands and expressed as percentage of siNC_Ctrl. β-actin was used as loading control. (D) mRNA LC3B and Smad2 fold expression levels quantified by qPCR analysis. ^†Denotes statistical significance when comparing TGFβ treatment versus control, using one-way ANOVA with multiple comparisons; ^‡denotes statistical significance when comparing TGFβ-treated siSmad2 versus the respective siNC treated control, using two-way ANOVA with Bonferroni post-test. *Denotes statistical significance compared to siNC control using t-test. Specific mean ± SD and p values, as well as densitometric analysis confirming knocked down of Smad2/3 are included in Supplemental Material. RPL: Relative protein levels.