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. 2009 Jan 12;184(1):67–82. doi: 10.1083/jcb.200801009

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© 2009 Zhang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — CDK6 and CDC25A are NANOG transcriptional targets. (A) Quantitative RT-PCR analysis for the expression of CDK6 and CDC25A in NANOG-overexpressing and control sublines. The value for the control clone from each cell line was set to 1, and all other values were calculated with respect to this. The data represent the mean ± SEM (error bars) from three independent experiments. (B) Quantitative RT-PCR analysis for the expression of NANOG, CDK6, and CDC25A in hESC 42 h after the transfection of NANOG siRNA. The data represent the mean ± SEM from three independent experiments (one in each cell line: H1, H9, and hES-NCL1). The value for the control siRNA samples was set to 1, and all other values were calculated with respect to this. (C) Western blotting for NANOG, CDK6, and CDC25A in hES-NCL1 transfected with NANOG or control siRNA 42 h after the transfection of NANOG siRNA. GAPDH is used as a loading control. Molecular masses are indicated in kilodaltons. (D and E) Bar chart showing enrichment of CDK6 intragenic DNA region (intron 1) and CDC25A promoter fragment after ChIP with NANOG antibody in hESCs and day-14 differentiated sample from EBs. The data represent the mean ± SEM (error bars) from two experiments performed in the H1 cell line. (F and G) Bar chart showing activation of CDK6- and CDC25A-luciferase constructs upon transfection of different domains of NANOG. For each reporter construct, luciferase activities relative to pGL4 promoterless controls were determined, and data are represented as the fold change caused by the indicated NANOG expression relative to the nonmutated reporter cotransfected with the empty (Flag) control. mCDK6 or mCDC25A indicates the CDK6- or CDC25A-luciferase constructs with a mutated NANOG-binding site. Flag, DNA construct without NANOG cDNA; ND + HD, DNA construct containing the homeodomain and the N-terminal region of NANOG; HD, DNA construct containing the homeodomain region of NANOG; HD + CD, DNA construct containing the homeodomain and C-terminal region of NANOG; Full length, full-length NANOG cDNA. In both panels, the luciferase activity for cells transfected with the Flag construct and CDK6-luciferase (F) or CDC25A-luciferase construct (G) was set to 1, and all other values, including the luciferase activity achieved with mutated CDK6 and CDC25A constructs, were calculated with respect to that. The data represent the mean ± SEM.