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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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Old passage E14 mESCs show differentiation defects and loss of methylation at ICRs. (a) Schematic representation of embryoid body (EB) differentiation protocol of YP- and OP- mESCs. (b) Representative bright field images showing EBs at day 4 (D4) and 9 (D9) obtained from both YP- and OP- E14 mESCs. Scale bar is 400 μm. (c) Quantitative real-time PCR showing the expression profiles of differentiation genes (Nkx2.5, Gata6, Otx2) and pluripotency genes (Rex1, Oct4, Nanog) in YP- and OP- E14 mESCs (ESC) and during EB differentiation at day 6 (D6) and day 12 (D12). (d) Schematic representation of neural differentiation protocol of YP- and OP- mESCs. (e) Quantitative real-time PCR showing the expression profiles of Sox1 at day 3 (D3) of N2B27 + retinoic acid (RA) treatment in YP- and OP-E14 mESCs (ESC). (f) Representative immunofluorescence images showing Nestin (left panels) and III β-tubulin (TUJ1, right panels) protein expression in YP- and OP- mESCs at day 8 (D8) of neural differentiation. (g) Quantitative real-time PCR experiment showing the expression profiles of Pax6 and Fgf5 at day 8 (D8) of N2B27 + retinoic acid (RA) treatment in YP- and OP- E14 mESCs (ESC). (c,e,g) 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 (c,e) and means of n = 3 technical replicated for SD (g). (c,e) Asterisks indicate statistical significance calculated by unpaired two-tailed t test analysis (n.s. not significant; *p < 0.05; **p < 0.01; ***p-value < 0.001). (h) Number of hypomethylated common CpGs in OP- versus YP- E14 mESCs analyzed by RRBS covered by at least 10 reads and showed at least 25% of methylation reduction. Red rectangle indicates imprinted regions. (i) Gene ontology of hypomethylated regions in OP-E14 mESCs analyzed by PANTHER (www.pantherdb.org). (j) Box-plot, from min–max values, showing the distribution of mCpG levels at ICRs in YP- and OP-E14 mESCs 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.