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. 2018 Nov 5;8:16338. doi: 10.1038/s41598-018-34612-3

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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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DNA demethylation and upregulation of Tgfb1 in mesangial cells of db/db mice. (A) Real-time PCR of Tgfb1 mRNA (normalized to Gapdh) in primary cultured mesangial cells from db/m and db/db mice. Open circle: The expression of Tgfb1 mRNA in db/db mice significantly higher than that in db/m mice. Data represent the mean ± SEM. n = 6. Filled circles: db/m mice; open circles: db/db mice. (B) Quantitation of DNA methylation (using a MethylCollector Ultra kit) in mesangial cells from 15-week-old db/m controls and db/db mice. Results of amplification from the −422 bp relative to the transcription start site (TSS) regions are shown. DNA methylation of db/db mice is significantly lower than that of db/m mice. n = 6. (C) Time course real-time PCR experiments demonstrating Tgfb1 mRNA (normalized to Gapdh) expression and quantitation of DNA methylation using a MethylCollector Ultra kit in 8-, 12-, and 15-week-old (8 W, 12 W, and 15 W, respectively) db/m and db/db mice. Following the significant difference in Tgfb1 mRNA at 8 week and the slight, but insignificant, decrease in DNA methylation in mesangial cells of db/db mice at 8 weeks, both parameters at 12 and 15 weeks significantly differed between db/m and db/db mice. n = 6. (D) ChIP analysis of DNMTs in mesangial cells of db/db mice. The results of ChIP assays for DNMT1, DNMT3A, and DNMT3B in mesangial cells from 12-week-old db/m and db/db mice at the positions 422 and 366 bp upstream of the TSS. The binding of DNMT1 and DNMT3B to the Tgfb1 promoter region is significantly decreased in db/db mice as compared to db/m mice, despite no difference of DNMT3A between both mouse strains. n = 6. (E) Real-time PCR of DNMTs mRNA (normalized to Gapdh) of DNMT1 in primary cultured mesangial cells from db/m and db/db mice. The expression of DNMT1 mRNA significantly and DNMT3b mRNA tends to decreased in db/db mice as compared to db/m mice. Data represent the mean ± SEM. real-time PCR; n = 6, Filled circles: db/m mice; open circles: db/db mice. (F) Western blot analysis of DNMT1 in primary cultured mesangial cells from db/m and db/db mice. Open circle: The expression of DNMT1 protein significantly decreased in db/db mice as compared to db/m mice. Data represent the mean ± SEM. n = 3. Filled circles: db/m mice; open circles: db/db mice. Original gel image of western blot analysis is presented in Supplemental Fig. 3.