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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 overexpressing mESC clones maintain high Wnt/β-catenin activity and normal ICR methylation level after several in vitro passages. (a) Scheme showing how β-catenin overexpressing mESC clones (S33Y #1, #2) were obtained and grown. (b) Representative FACS-plot showing the percentage of positive mESCs for 7TGP topflash reporter activity in YP-mESCs, OP-mESCs, OP-S33Y #1 and OP-S33Y #2 mESC clones. The non infected (NI) cells were used as negative control (Ctrl-). The FITC and the Per-CP-Cy5.5-A detectors were used to identify GFP +(y axis) and autofluorescence (false positive) cells (x axis). The number of recorded events spans from 12000 (OP-mESC) to 18000 (YP-mESCs). Data are represented as means of n = 3 independent experiments ± SD. (c) Quantitative real-time PCR experiments showing the expression profiles of Axin2 in YP-mESCs, OP-mESCs, OP-S33Y #1 and OP-S33Y #2 mESC clones. The transcriptional levels are normalized to Gapdh as a reference gene. Data are represented as fold change (2^−ΔΔCt) relative to the YP-E14 mESCs and the results 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 < 0.001). (d) Western blot analysis showing total β-catenin protein levels in E14 YP-mESCs, OP-mESCs, OP-S33Y #1 and OP-S33Y #2 mESC clones and its quantification. Data are represented as fold change over the protein amount in YP-mESCs and means of n = 3 independent experiments ± SE. β-tubulin was used as loading control. 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. (e) Cluster analysis of the four different mESCs. For each line 2 different biological replicates were represented. (f) Box-plot, from min–max values, showing the distribution of mCpG levels at ICRs in OP-S33Y #1 and OP-S33Y #2 mESC clones determined by RRBS analysis. The plots indicate the first quartile, median (black line) and third quartile. Data are obtained from the average of n = 2 biological replicates. (g) Heat-map representation of ICR methylation levels in YP-mESCs, OP-mESCs, OP-S33Y #1 and OP-S33Y #2 mESC clones.