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. 2019 Jan 30;9:948. doi: 10.1038/s41598-018-37442-5

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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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β-catenin silencing impairs mESC differentiation. (a) Quantitative real-time PCR analysis showing β-catenin silencing efficiency, Axin2 and pluripotency marker (Nanog, Oct4) levels. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2^−ΔΔCt) relative to the shCtrl-infected mESCs and the results are means of n = 3 technical replicated ± SD. (b) Western blot analysis showing protein levels of total and nuclear β-catenin in shCtrl-, shβcat#1-, shβcat#2- and shβcat#3- transduced E14 mESCs (n = 1). Quantification of total and nuclear β-catenin is represented as relative to total β-catenin in shCtrl-transduced mESCs. β-tubulin and H3 were used as loading controls. For western-blot quantification densitometric analysis was carried out by using ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. (c) Representative bright field images of mESCs and embryoid bodies (EBs) at day 3 (D3), 8 (D8) after β-catenin silencing (shβcat #1, #2, #3) versus the control condition (shCtrl). Scale bar is 400 μm. (d) Quantitative real-time PCR experiments showing the expression profiles of ERVs (IAP, MusD, MERVL) in shCtrl-, shβcat#1-, shβcat#2- and shβcat#3- transduced E14 mESCs. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2^−ΔΔCt) relative to shCtrl-infected mESCs and are means of n = 3 independent experiments ± SE. Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p < 0.05; **p < 0.01). (e) Schematic representation showing cellular and epigenetic changes occurring in prolonged in vitro mESC cultures along with Wnt/β-catenin pathway downregulation.