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. 2020 Nov 27;11:6053. doi: 10.1038/s41467-020-19879-3

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© The Author(s) 2020

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. 4 — a Examples of nuclei after H3K27me3 immunostaining (red) and Hoechst 33342 staining (blue) in WT, ΔFirre^Xa, and ΔFirre^Xa transfected with a mouse Firre transgene (ΔFirre^{Xa+mtransgene}) or a human FIRRE transgene (ΔFirre^{Xa+htransgene}). b The bar plot shows a significantly higher percentage of nuclei with a H3K27me3 cluster in cells with a mouse or human Firre/FIRRE transgene, compared to ΔFirre^Xa (p value = 2.8075e-16 and 0.0000134599, respectively). c Examples of nuclei after RNA-FISH for Xist (green) and Firre (red) in a ΔFirre^{Xa+mtransgene} cell clone with high expression of the mouse transgene. The upper two nuclei show association between Firre and Xist signals (seen in 15% of nuclei), and the lower nuclei, lack of association. d The bar plot shows the percentage of nuclei with a H3K27me3 cluster in MEFs derived from Firre^+/− and Firre^−/− females that harbor a doxycycline (Dox) inducible transgene (Firre^+/− tg;rtTA; -Dox; Firre^+/− tg;rtTA; +Dox; Firre^−/− tg;rtTA; -Dox; Firre^−/− tg;rtTA; +Dox), compared to WT and mutants. The percentage of nuclei with a H3K27me3 cluster increases significantly in Firre^+/− tg;rtTA and Firre^−/− tg;rtTA MEFs after addition of doxycycline (Dox+) (p value=4.31567e-30 and 3.51193e-20, respectively). A total of >300 nuclei scored for the presence of a H3K27me3 cluster (a, b, d), or for Xist and Firre RNA-FISH signals (c) per cell type over three independent experiments; significance was determined by one-sided Fisher exact test; bar plots are presented as mean values ± SEM; scale bars represent 10 µm.