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. 2018 Sep 11;9(9):924. doi: 10.1038/s41419-018-0991-1

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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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Fig. 7 — a Protein levels of pSmad1/5 in WT ESCs and four Cited1 KO ESC clones after treatment with BMP4 for 1 day and 6 days. b Co-IP assay to show that exogenously expressed Flag-Cited1 binds to endogenous Smad4 specifically in E14T ESCs. Whole-cell lysates (WCL) were immunoprecipitated with anti-Flag M2 beads and analyzed by western blotting with anti-Smad4 and anti-Flag antibodies, respectively. Five percent of the WCL was loaded as the input. c qRT-PCR analysis of transcript levels of trophoblast specific markers in control E14T ESCs (shRNA_ NT) or p300 KD cells (p300 shRNA#5, 6, 8) upon Cited1 overexpression for 3 days. The average mRNA level in cells transfected with an empty vector (pPy) and a non-targeting shRNA plasmid (shRNA_NT) was set at 1.0. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01. d A proposed working model for Cited1 to promote mouse ESC trans-differentiation toward a trophoblast-like state through activation of BMP signaling. On the one hand, Cited1 binds to Bmpr2 to enhance the phosphorylation of Smad1/5/8. On the other hand, Cited1 may functionally link Smad4 and p300 to activate trophoblast trans-differentiation associated transcriptional program