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. 2014 Oct 15;28(20):2219–2232. doi: 10.1101/gad.247163.114

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© 2014 Rhee et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 3. — Induction of Arid3a converts ES cells to stable TS-like cells that can further differentiate to trophoblast lineages. (A) Cell morphologies of control ES and Arid3a-overexpressing cells in TS cell derivation medium containing Fgf4 and heparin. The cells were plated at clonal density, cultured for 5 d, and stained for AP. (B) Expression analysis of TE markers (red) by RT-qPCR in Arid3a-overexpressing cells in TS cell derivation medium (gray) and TS cells (white). The data are plotted relative to the corresponding expression levels of control ES cells grown in TS cell derivation medium. (C) Cell morphologies of Arid3a-overexpressing cells adapted to TS cell derivation medium (top) followed by removal of Fgf4 and heparin (bottom). Arrows indicate TGCs. (D) Arid3a OE in TS cell differentiation conditions promotes differentiation to more specialized cells of the TE lineage. Expression analysis of TE and trophoblast markers (red) as determined by RT-qPCR in Arid3a-overexpressing cells adapted to TS cell medium upon withdrawal of Fgf4 and heparin (gray). The data are plotted relative to corresponding expression levels of Arid3a-overexpressing cells adapted to TS cell culture medium (white). (E) Cell morphologies upon knockdown (KD) of Oct4 and double knockdown (dKD) of Oct4 + Arid3a or Oct4 + Cdx2 in ES cells under normal ES culture conditions. (F) Expressions of TE (red) and pluripotency-associated (green) genes as determined by RT-qPCR following the indicated knockdown perturbations (knockdown or double knockdown) in ES cells (LIF⁺ conditions). The data are plotted relative to the corresponding expression levels in Oct4 knockdown cells. (G) Cell morphologies of control TS and Arid3a-overexpressing TS cells under TS cell derivation conditions. Arrows indicate TGCs. (H) Expression analysis of TE differentiation markers (red) upon OE of Arid3a in TS cells (gray) and in control TS cells (white) by RT-qPCR. Error bars depict standard deviations of biological triplicates.