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. 2016 Nov 8;35(3):611–625. doi: 10.1002/stem.2521

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© 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Pluripotency genes are not efficiently downregulated during differentiation in Zeb2 knockout (KO) embryonic stem cells (ESCs). (A): Control (Ctrl) and Zeb2 KO (KO) embryoid bodies (EBs) stained for Nanog (brown) on d4, d6, and d15 of neural differentiation (ND). (B): Ctrl and KO EBs costained for Oct4 (green) and Cdh1 (red) on d4, 6, and 15 of ND. Panels A, B show results from one experiment that is representative for three experiments. Scale bar: 50 µm. (C, D): Zeb2 chromatin immunoprecipitation (using anti‐Flag antibody) on Cdh1 (panel C) and Nanog promoter (panel D). Results shown are from one experiment and representative for three experiments. SD of two technical repeats is shown. (E, F): Ctrl and KO ESCs subjected to ND (panel E) and general differentiation (panel F) for 15 days, dissociated and plated at 500 cells/well in 2i. The resulting ESC colonies (indicated by arrows) were visualized by staining for AP, and panel (G) represents the average number of AP+ colonies obtained after plating the cells. Abbreviations: ND, neural differentiation; GD, general differentiation.