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. 2018 Jul 4;8:10090. doi: 10.1038/s41598-018-28480-0

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© The Author(s) 2018

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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Relative DIO2 mRNA expression levels in cAMP-treated cells and cAMP assay results in TSH-treated in primary-cultured skin fibroblast cells. (a) Cells are treated with 1 mM db-cAMP for 16 h (+cAMP) and total RNA is extracted from the cells and DIO2 mRNA expression level is analyzed by quantitative real-time RT-PCR. The cAMP-non-treated cells are used as negative control (−cAMP). DIO2 mRNA expressions in WT, Hetero, Homo and patient are significantly increased by cAMP treatment. *p < 0.05 vs. negative control in each tested fibroblast. (b) Cells were treated with 1 IU of TSH for 30 min and then are treated with 0.1 N hydrochloric acid to stop endogenous phosphodiesterase activity. After cell lysis, intracellular cAMP concentration is measured using cAMP direct immunoassay kit. The TSH-non-treated cells are used as negative control. cAMP is increased in all groups, but significantly less increased in the patient. *p < 0.05 vs. negative control in each tested fibroblast. ^#p < 0.05 vs. WT.