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. 2017 Aug 15;7:8277. doi: 10.1038/s41598-017-08883-1

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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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SIRT1 defect increased pMKK3/6 by increasing MKK6 levels. The SIRT1 and MKK6 mRNA (A) and protein (B) expression levels in CT-and PT-ECFC were assessed via RT-qPCR and Western blot analysis, respectively. (A) The impact of PT on the mRNA level (relative expression to CT; N = 13 CT vs. 16 PT). (B) The impact of PT on the protein level (relative expression to actin; N = 18 CT vs. 26 PT, representative blot). (C) The correlation between MKK6 and SIRT1 protein expression levels. Every plot represents a single sample collected from a patient (N = 24). (D) Western blot analysis of MKK3/6 activation in CT- and PT-ECFC was performed by the determination of phosphor-MKK3/6, MKK3 and MKK6 (N = 118 vs 28). (E–J) The impact of SIRT1 modulation on the activation of MKK3/6. SIRT1 and MKK6 mRNA (E–G) and protein (H–J) expression levels, along with MKK3/6 phosphorylation, were assessed via RT-qPCR and Western blot analysis, respectively. (E,H) The impact of SIRT1 silencing on the mRNA and protein expression of CT-ECFC (relative expression to scrambled; N = 12). (F,I) The impact of SIRT1 overexpression on mRNA and protein expression in PT-ECFC (relative expression to empty; N = 7 and 11, respectively). (G,J) The impact of NAM on mRNA (relative expression to empty, N = 8) and protein expression (relative expression to SIRT1-transfected cells; N = −6–9) in PT-ECFC overexpressing SIRT1. The data for all of the bar graphs are presented as the means ± SEM. (Statistical analysis: t-test; *p < 0.05, **p < 0.01, ***p < 0.001). All blots correspond to representative images of blot bands.