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. 2020 Dec 15;40(6):1191–1202. doi: 10.1038/s41388-020-01591-7

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© The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature 2020

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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Fig. 2 — A Western blot analysis of G9a, H3K9me2, and dot blot analysis of 5-methylcytosine (5meC: methylated DNA) levels in G9a knockdown t-hESCs vs. scramble control shRNA (shCTRL). GAPDH was used as loading control. Relative OD signal quantification vs. GAPDH intensity are presented (n = 3, ***: p = 0.0001; **: p ≤ 0.0057). B Growth rate assessment of shG9a vs. shCTRL t-hESC cultures. Cell counts were acquired at 24 h (*: p = 0.021), 48 h (*: p = 0.034), and 72 h (***: p = 0.0003) post-seeding (30 K cells/well, n = 3). C Testicular teratoma assay using shCTRL (n = 5) and shG9a (n = 3) t-hESCs over 14 days. 1 × 10⁵ cells (left testicle) or saline (right testicle) were injected in each mouse. Tumor formation frequencies and tumor/control size ratios were presented for both groups (*: p = 0.043). D Western blot analysis of H3K9me2 and dot blot analysis of 5 meC levels upon pharmacological inhibition of G9a HMTase activity (BIX-01294: 1 μM, UNC0642: 5 μM, 48 h) in t-hESCs (H3K9me2: n ≥ 4, **: p = 0.0067; *: p = 0.031; 5 meC: n ≥ 3, ***: p = 0.00057; **: p = 0.0083). E Methylated DNA immunoprecipitation (MeDIP) assay showing variations of methylation levels in t-hESCs LINE transposable elements upon BIX-01294 (1 μM) and UNC0642 (5 μM) treatments, relative to DMSO controls (total LINEs: n = 3, **: 0.0082; *: p = 0.013; LINE-1 ORF2: n = 5, ***: p = 0.0004; **: p = 0.0072; LINE-1 5′-UTR: n = 6, ***: p ≤ 0.00089). Methylation of endogenous retroviral elements hERV-H was not affected by G9a inhibition (n = 6). F mRNAsi from RNA-seq transcriptome profiling was determined for BIX-01294 (1 μM) and DMSO treated t-hESCs. G GSEA revealed negative correlations between significant transcriptional modulations induced by BIX-01294 in t-hESCs (vs. DMSO, p < 0.05) and genes upregulated in embryonic stem cell, as well as genes induced by MYC. H Heat maps representing expression of significantly modulated genes (p < 0.05) associated with cell cycle (Negative Reg.: GO:0045786, Positive Reg.: GO:0045787) and differentiation regulation (Positive Reg./Stem Cell Diff.: GO:0045597, GO:0048863, Negative Reg.: GO:0045596) in BIX-01294-treated t-hESCs vs. DMSO controls.