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. 2015 Nov 28;44(6):2613–2627. doi: 10.1093/nar/gkv1315

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — LncRNA Gm15055 is maintained by OCT4 in mESCs. (A) Gm15055 expression in mESCs, E8.5 and E9.5 mouse tailbuds, E14.5 mouse yolk sac and MEF cells was quantified by RT-qPCR. The expression levels are shown relative to that of mESCs; Gapdh was used as an internal control. (B) The relative expression of Gm15055 and Oct4 during embryoid body differentiation of mESCs was detected by RT-qPCR at the indicated time points. (C) RT-qPCR detection of the Oct4 knockdown efficiency and the resulting Gm15055 expression in mESC. Two siRNAs targeting mOct4 (SiOct4-1 and SiOct4-2) and one control siRNA (SiNC) were used, with two different amounts of siRNA as indicated. (D) The pluripotency factors Oct4, Sox2, Nanog were overexpressed separately or in combination in MEF cells and the expression level of the endogenous Gm15055 was detected by RT-qPCR. GFP overexpression was used as a control. +OS: +Oct4+Sox2; +OSN: +Oct4+Sox2+Nanog. (E) Recruitment of OCT4 at the Gm15055 locus was analyzed by ChIP-qPCR. Three potential OCT4 binding regions at the Gm15055 locus (namely OBR 1, OBR 2 and OBR 3) and the Gm15055 promoter (GmP) were examined. (F) The activity of OCT4 on Gm15055 promoter with or without the downstream OCT4 binding regions were detected in reporter assay in 293T cells. The pGL3 basic (Basic), Gm15055 promoter-pGL3 (GmP) or OCT4 binding region 2/3-Gm15055 promoter-pGL3 (OBR 2/3-GmP) reporters, was co-transfected with empty or Oct4 overexpression vector, respectively. The relative luciferase activity was normalized to that of the Basic plus empty overexpression (OE empty) group. (G) Reporter assay in 293T cells showed that mutation to the OCT4 binding sites abolished the response of Gm15055 promoter to the OCT4 activity. The pGL3 basic (Basic), Gm15055 promoter-pGL3 (GmP) or that carrying single/double mutations at the Gm15055 promoter (GmP mut1, GmP mut2 and GmP mut1+2), was co-transfected with empty or Oct4 overexpression vector, respectively. The relative luciferase activity was normalized to that of the Basic plus empty overexpression (OE empty) group.