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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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Prolonged in vitro cell culture of E14 mouse embryonic stem cells (mESCs) correlates with low Wnt/β-catenin activity. (a) Schematic representation of Young (YP) and Old passage (OP) E14 mESCs. (b) Representative bright field images of YP- and OP-mESCs. Round-shaped and flat colonies are indicated by white and yellow arrow, respectively. (c) Quantitative real-time PCR showing the expression profiles of Axin2, Lef1, Tcf1, Sp5 in YP- and OP- mESCs. The transcriptional levels are normalized to Gapdh as reference gene. Data are represented as fold change (2^−ΔΔCt) relative to the YP-E14 mESCs and means of n = 3 independent experiments ± SE. (d,e) Representative immunofluorescence (d) and confocal microphotographs (e) of β-catenin. Nuclear demarcation is indicated by white circles (right panel). (f) Western blot analysis showing total and nuclear β-catenin protein in YP- and OP-mESCs and its quantification (n = 1) relative to total β-catenin in YP-mESCs. For quantification, densitometric analysis was performed with ImageJ software. The quantification reflects the relative amounts as a ratio of each protein band relative to their loading control. (g) Representative immunofluorescence images showing OCT4 (green), NANOG (red) and their merge in YP- and OP-E14 mESCs. (h,i) Representative western blot analysis of OCT4 and NANOG in YP- and OP-mESCs (h) and its quantification represented as fold change over the protein amount in YP-mESCs and means of n = 3 independent experiments ± SE (i). (f,h,i) Full scan blots are available in Supplementary Fig. 6. (j–m) FACS-plot showing the percentage of E-cadherin +(j) and SSEA1 +cells (l) in YP- and OP- mESCs and its quantification (k,m) as means of 3 technical replicates ± SE (NS: non stained). (n,o) Representative cell cycle FACS profile analyzed with Flowjo software (n) and its quantification (o) represented as percentage of total cells and means of n = 3 independent experiments ± SE. Scale bar is 200 (b,d,g) and 10 μm (e). (d,e left panel, and g) Nuclei were stained with DAPI. (f,h) β-tubulin and H3 were used as loading controls. (c,i,k,m,o) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p-value < 0.05; ***p-value < 0.001).