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. 2017 Apr 11;7:812. doi: 10.1038/s41598-017-00845-x

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© The Author(s) 2017

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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TGF-β induces an increase in levels of PTEN and its phosphorylation in human TM cells. (A,B) Real-time PCR analysis of COL1A1 and PTEN mRNA levels in TM cells treated with TGF-β2 (1 or 5 ng/ml). RNA was extracted at 12 and 24 h. mRNA expression levels were normalized to EUK18S. A two-way ANOVA with Bonferroni’s post-hoc was used to determine statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001. N = 3. (C,D) Western blot analysis of collagen levels in TM cells treated with TGF-β (T1, T5, T10 indicates 1, 5 or 10 ng/ml TGF-β2). Protein was extracted at 24 and 48 h. TGF-β receptor signaling was inhibited using the inhibitor SB431542 (SB; 20 μM). D7278- representative human donor. (E,F) Western blot analysis of PTEN levels and its phosphorylation (pPTEN- ser380/thr382/383) in TM cells treated with TGF-β and SB431542. Protein was extracted at 24 and 48 h. D7278- representative human donor. (G,H) Densitometry was performed on PTEN and pPTEN Western blot data at 48 h using ImageJ software. Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc. *p < 0.05, **p < 0.01, ***p < 0.001. N = 3. (I,J). TGF-β-mediated induction of PTEN levels and its phosphorylation was confirmed in TM cells from an adult human donor, aged 26 yrs (Adult 26Y).