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. 2019 Jul 1;33(13-14):782–798. doi: 10.1101/gad.325696.119

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© 2019 Schüle et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — Gadd45 TKO ESCs show impairment of the 2C-like state. (A) Enrichment analysis of genes down-regulated in Gadd45 TKO ESCs using the ESCAPE database (Xu et al. 2014). Enrichment analysis shows a significant overlap with genes deregulated (up or down) upon overexpression of, for example, Gadd45a or Zscan4c. (B) Scatter plot of the common deregulated genes (gray) in Gadd45 TKO ESCs and up-regulated in the 2C-like state (log₂FC [log₂ of fold change versus control ESCs]) (Macfarlan et al. 2012). (C) Overlap between 2C-associated (Macfarlan et al. 2012) and hyper-DMR-associated genes. (D) qPCR expression analysis of selected 2C-associated genes and retroviral elements in control (Co) and Gadd45 TKO ESCs 48 h after transfection with the indicated genes. Expression is relative to GFP transfected control ESCs. Data are presented as mean ± SD from n = 3 independent clones and n = 2 independent experiments. Statistical significance was tested with two-tailed paired Student's t-test. (E) Scatter plot showing Zscan4c::eGFP-positive cells in bulk control ESCs measured by flow cytometry analysis. Representative gates used for bulk analysis are boxed. (F) Flow cytometry analysis of Zscan4c::eGFP-positive cells in control and Gadd45 TKO ESCs. (G) Flow cytometry analysis of Zscan4c::eGFP-positive cells in control or Gadd45-TKO ESCs 48 h after transfection with the indicated genes. Data are presented as means ± SD from n = 3 independent clones and n = 2 independent experiments. Statistical significance was tested with two-tailed unpaired (TKO vs. control [Co]) or paired (BFP vs. Gadd45 overexpression) Student's t-test. (H) Flow cytometry analysis of Zscan4::eGFP-positive cells in control ESCs 48 h after transfection with the indicated genes. Statistical significance was tested with two-tailed paired Student's t-test. (I,J) 5mC and 5hmC levels in unsorted or Zscan4⁻ or Zscan4⁺ sorted control ESCs and Gadd45 TKO ESC clones determined by LC-MS/MS. Values are the percentage of total cytosine (C).