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. 2022 May 6;13:2516. doi: 10.1038/s41467-022-30259-x

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© The Author(s) 2022

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. 9 — a Strategy for the differentiation of mESC lines into mEpiLCs and culture of mEpiLCs with and without the GSK-3 inhibitor CHIR99021 (CHIR; 3 μM). b Representative images of mEpiLCs with high, intermediate, and low percent of nuclei with Xist RNA coating (green). Nuclei are stained blue with DAPI. At least ten nuclei were counted per colony in mEpiLC RNA FISH quantification. Scale bars are ~100 microns. c Percentage of nuclei with Xist RNA coating in differentiating mEpiLCs with and without CHIR generated from three independent ESC lines. mEpiLCs cultured with CHIR lost a significant proportion of Xist RNA coating compared to mEpiLCs cultured without CHIR in all three mEpiLC replicates (general linear model comparison, p < 0.001). At least ten nuclei were counted per colony for mEpiLC RNA FISH quantification. The total number of colonies quantified at each passage range between 12 and 20 and are cataloged in source data. Source data are provided as a Source Data file.