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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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The effect of treatment with the anti-oxidant, Tempol to demethylauin of Tgfb1 in db/db mice and reactive oxygen species to methylation of Tgfb1 in mesangial cells. (A) Twenty-four hour urine samples were collected from db/m, db/db, and db/db mice treated with Tempol for 8 weeks in metabolic cages, and 8-OHdG was measured using a New 8-OHdG Check ELISA kit. Immunoblotting of MAD in whole kidneys of db/m, db/db, and db/db mice treated with Tempol for 8 weeks. Urinary 8-OHdG and renal MAD content were significantly increased in db/db mice compared with db/m mice, but treatment with Tempol significantly decreased these parameters. Beta-actin was used for normalization. Data represent the mean ± SEM. 8-OHdG, n = 4; MAD, n = 3. Filled circle: db/m mice; open circle: db/db mice; open triangle: db/db+ Tempol. (B) DNA methylation and down-regulation of Tgfb1 in mesangial cells from db/db mice treated with Tempol. Quantitation of DNA methylation, using a MethylCollector Ultra kit, in mesangial cells from 15-week-old db/db mice, treated or not treated with Tempol for 8 weeks. Quantitative real-time PCR of Tgfb1 and Gapdh mRNA from 15-week-old db/db mice treated or not treated with Tempol for 8 weeks. Tempol significantly increase DNA methylation and decreased Tgfb1 mRNA. Data represent the mean ± SEM. n = 6. (C) The results of ChIP assays to determine binding of DNMT1 and USF1 to the Tgfb1 promoter (TSS −641 to −636) in mesangial cells from 15-week-old db/db mice treated or not treated with Tempol for 8 weeks. Tempol significantly increase and decrease the binding of DNMT1 and USF1 to their respective binding sites. Data represent the mean ± SEM. n = 6. (D) Quantitation of DNA methylation, using a MethylCollector Ultra kit, in mesangial cells from 8-week-old m/m mice treated with 0 (Vehicle) or 60 µM H₂O₂. Quantitative real-time PCR of Tgfb1 and Gapdh mRNA in mesangial cells from 8-week-old m/m mice treated with 0 or 60 µM H₂O₂. Treatment with H₂O₂ significantly decrease DNA methylation and increase Tgfb1 mRNA expression. Data represent ± SEM. n = 6. (E) The effect of siRNA targeting Usf1 on upregulation of Tgfb1 mRNA by reactive oxygen species. Quantitative real-time PCR of Tgfb1 (normalized to Gapdh) mRNA in mesangial cells, from 8-week-old m/m mice, treated with 0 or 60 µM H₂O₂, and with control or Usf1-targeting siRNA. Transfection of Usf1 siRNA abolish the upregulation of Tgfb1 mRNA induced by H₂O₂, whereas control siRNA did not affect H₂O_2-induced increase in Tgfb1 mRNA_. Dunnett -corrected t-tests. Data represent the mean ± SEM. n = 6. (F) Quantitative real-time PCR of DNMT1 mRNA (normalized to Gapdh) in mesangial cells, from 8-week-old m/m mice, treated with 0 or 60 µM H₂O₂. H₂O₂.induced significant decrease in DNMT1 mRNA. Data represent the mean ± SEM. n = 6. (G) Quantitative real-time PCR of DNMT1 mRNA (normalized to Gapdh) and western blot analysis of DNMT1 (normalized to β-actin) from 15-week old db/db mice treated or not treated with Tempol for 8 weeks. Tempol tended to increase DNMT1mRNA and protein. Data represent the mean ± SEM. real-time PCR; n = 6, western blot analysis; n = 3. Original gel image of western blot analysis are presented in Supplemental Fig. 9.