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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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Bisulfite sequence analysis of the Tgfb1 promoter region, and ChIP assays of USF1 and SREBP1. (A) Bisulfite sequence analysis of the Tgfb1 promoter region in mesangial cells from 15-week-old db/m and db/db mice. Top, schema of the Tgfb1 promoter. The dashes and numbers indicate the positions of the cytosine residues of CpG dinucleotides relative to the transcription start site (TSS, +1). Bottom, DNA methylation status of the CpG sites between −207 and −685 bp relative to the TSS. CpGs at positions 639, 458, 422, and 406 bp upstream of the TSS are demethylated in db/db mice, while CpGs at positions −366 and −207 were comparably demethylated in both mouse strains. Filled circles represent methylated and open circles demethylated CpG sites. (B) The results of ChIP assays of USF1 binding to the site, TSS −641 to −636, and SREBP1 binding to the site, TSS −200 to −32, in mesangial cells from 12-week-old db/m and db/db mice. The binding of USF1 to its recognition site in Tgfb1 promoter region (−641 to −636 bp) is significantly increased in db/db mice compared with db/m mice, but the binding of SREBP1 to its recognition site (−232 to −220 bp) is comparable between db/m and db/db mice. Data represent the mean ± SEM. n = 6. Filled circles: db/m mice; open circles: db/db mice. (C) The results of ChIP assays of DNMT1 and DNMT3B binding to the USF1 recognition site (TSS −641 to −636) in mesangial cells from 12-week-old db/m and db/db mice. The binding of DNMT1 and DNMT3B to the corresponding promoter region are significantly decreased in db/db mice; the greater binding of DNMT1 than that of DNMT3B. Data represent the mean ± SEM. n = 6. (D) Effect of folic acid on DNA methylation and Tgfb1 mRNA in mesangial cells. Quantitation of DNA methylation in mesangial cells from 15-week-old db/db and db/db mice treated with Folic acid for 8 weeks revealed tha DNA methylation of db/db mice with Folic acid is significantly higher than that of db/m mice. Results of amplification from the −639 bp relative to the transcription start site (TSS) regions are shown. Real-time PCR of Tgfb1 mRNA (normalized to Gapdh) in primary cultured mesangial cells from db/db and db/db mice treated with Folic acid. The expression of Tgfb1 mRNA in db/db mice with Folic acid was significantly lower than that in db/db mice. Data represent the mean ± SEM. n = 6. Open circles: db/db mice; open triangles: db/db mice treated with Folic acid. (G) Effect of folic acid on DNMT1 mRNA in mesangial cells. Real-time PCR of DNMT1 mRNA (normalized to Gapdh) in primary cultured mesangial cells from db/db and db/db mice treated with Folic acid. The expression of Tgfb1 mRNA in db/db mice with Folic acid was significantly higher than that in db/db mice. Data represent the mean ± SEM. n = 6. Open circles: db/db mice; open triangles: db/db mice treated with Folic acid.