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. 2019 Mar 7;9:3831. doi: 10.1038/s41598-019-40358-3

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© The Author(s) 2019

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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Iws1 is required for the expression of Nanog. (A) EU-derived fluorescence was used to assess the global transcription in embryos electroporated with the indicated siRNAs. The fluorescence intensity of EU is highest in 2-cell stage siControl embryos and exhibits drastic decreases in siIws1 and siSupt6 embryos at 96 hphCG. At least 20 embryos were used for each experiment. (B) Immunofluorescent detection of Pou5f1 (Oct4), Cdx2 and Nanog in siControl, siIws1 and siSupt6 embryos. Representative images of E3.5 embryos are shown. DNA was counterstained with DAPI. At least 30 embryos were used for each experiment. (C) RT-qPCR analysis of the Pou5f1, Cdx2 and Nanog mRNA in siControl, siIws1 and siSupt6 embryos at the indicated times after hCG injection. The mRNA expression of H2afz was used as an internal control. The mRNA expression levels of Pou5f1, Cdx2 and Nanog in siControl embryos at 44 hr, 70 hr, and 44 hr, respectively, were defined as 1. One-hundred embryos were used for each analysis. (D) PLA showing the apparent interaction of Prmt5 and Iws1 in the cell nuclei of representative 4-cell stage embryos. (E) PLA showing that Prmt5 and Nanog interact in early embryos and that Iws1 is required for this interaction. DNA was counterstained with DAPI. At least 20 embryos were used for each experiment. Scale bars: 20 μm.