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. 2015 Jan 8;6(1):e1588. doi: 10.1038/cddis.2014.551

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Copyright © 2015 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0

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Oct4 positively regulates Rif1 and is indispensable for Smad3 to bind to Rif1 promoter regions. (a) Quantitative real-time PCR to examine the mRNA levels of Pou5f1 and Rif1 after transfection with pSuper control and pSuper-Pou5f1-shRNA plasmids. Actin was analyzed as a control. The data are shown as the mean±S.D. (n=3). (b) Luciferase assay to examine Rif1 promoter activity in mouse ESCs transfected with pSuper control and pSuper-Pou5f1-shRNA plasmids. Renilla was analyzed as an internal control. The data are shown as the mean±S.D. (n=3). (c) Quantitative real-time PCR to examine the mRNA levels of Pou5f1, Lefty1 and Rif1 after transfection with pCAG-GFP and pCAG-Pou5f1 plasmids. Actin was analyzed as a control. The data are shown as the mean±S.D. (n=3). (d) Western blot analysis of the protein levels of Oct4 and Rif1 in mouse ESCs transfected with pCAG-GFP and pCAG-Pou5f1 plasmids. Gapdh was analyzed as a control. Arrow indicates the overexpression band of Flag-Oct4. (e) ChIP-qPCR to examine the DNA enrichment of Oct4 and control IgG at the Smad3-binding sites on the promoter of Rif1 in mouse ESCs. Enrichment of studied proteins on Actin was analyzed as a control. The data are shown as the mean±S.D. (n=3). (f) ChIP-qPCR to examine the Oct4 enrichment at the Lefty1, Lefty2 and Rif1 in WT and Smad3−/− ESCs. Actin was analyzed as a control. The data are shown as the mean±S.D. (n=3). (g) ChIP-qPCR to examine Smad3 enrichment at the Lefty1, Lefty2 and Rif1 at 1-day puromycin selection after mouse ESCs were transfected with pSuper control and pSuper-Pou5f1-shRNA plasmids. Smad3 enrichment at Actin was analyzed as a control. The data are shown as the mean±S.D. (n=3). (h) Sequential ChIP assay was performed to examine Smad3 and IgG enrichment on Oct4-enriched DNAs. The quantity of enriched Lefty1, Lefty2 and Rif1 (Rif1-1 and Rif1-2) fragments was checked by real-time PCR. The data are shown as the mean±S.D. (n=3). Statistically significant differences, calculated through student's t-tests, are indicated (*P<0.05; **P<0.01; ***P<0.001)