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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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Smad3 represses Rif1 expression in ESCs. (a) The heat map shows the expression profile of genes with mRNA level increased or decreased for more than 1.5-fold in Smad3−/− ESCs compared with WT ESCs. Lefty1 and Smad3 are decreased, whereas Rif1 is increased in Smad3−/− ESCs compared with WT ESCs. (b) Quantitative real-time PCR to examine the mRNA level of Rif1 in WT and Smad3−/− ESCs. Actin was analyzed as an internal control. The data are shown as the mean±S.D. (n=3). (c) Western blot and densitometric analyses of the expression of Rif1 in WT and Smad3−/− ESCs. Gapdh expression level was used as an internal control. The data are shown as the mean±S.D. (n=2). (d) Western blot analysis of Smad3 (upper layer) and real-time PCR analysis of Lefty1 and Rif1 (lower layer) in pCAG-GFP- and pCAG-Smad3-transfected Smad3−/− ESCs. Gapdh protein level and Actin expression level were used as internal controls for the western blot and real-time PCR analysis, respectively. Arrow indicates the overexpression band of Flag-Smad3. The real-time PCR data are shown as the mean±S.D. (n=3). (e) Quantitative real-time PCR to examine mRNA expression levels of Lefty1, Lefty2 and Rif1 in mouse ESCs with Activin A (25 ng/ml) treatment for 0 and 24 h. Actin was analyzed as an internal control. The data are shown as the mean±S.D. (n=3). (f) Quantitative real-time PCR to examine mRNA expression levels of Lefty1, Lefty2 and Rif1 in mouse ESCs with SB431542 (10 μM) treatment for 0 and 24 h. Actin was analyzed as an internal control. The data are shown as the mean±S.D. (n=3). (g) ChIP-qPCR to examine Smad3 and IgG enrichment on the promoter of Rif1. The sketch of Smad3-binding sites on the promoter of Rif1 has been indicated (SBS1 and SBS2), Rif1-1 and Rif1-2 regions cover SBS1 and SBS2, respectively. Protein enrichment on Actin was analyzed as a control. The data are shown as the mean±S.D. (n=3). (h) Luciferase assay to examine Rif1 promoter activity in WT and Smad3−/− ESCs at 48 h after transfection. Two kb Rif1 promoter was cloned in front of firefly luciferase reporter. Renilla was analyzed as an internal control. 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)