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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. 3 — A GSEA showing a negative correlation between genes significantly modulated by BIX-01294 in t-hESCs (vs. DMSO, p < 0.05) and genes upregulated in human CRCs. B Hierarchal clustering analysis of BIX-01294-treated t-hESC and HCT116 cells, as well as COAD samples (vs. normal tissues) based on CRC upregulated genes. C Candidate-based validation of Grade CRC upregulated (M16740) genes upon G9a inhibition using UNC0642 (2.5 μM, 48 h) in HCT116 cells (n = 3, ***: p = 0.0006; **: p = 0.0045; *: p ≤ 0.028). D Dose-response curves assessing selective toxicity (Sel-Tox) of G9a inhibition (BIX-01294) in t-hESCs vs. normal human ES cells (hESC), as well as cancer stem-like HT29 vs. normal HIEC cells (BIX-01294 and UNC0642). Sel-Tox ratios were determined as follow: EC₅₀Normal/EC₅₀Cancer (n = 3). E Growth rate assessment of G9a knockdown (shG9a, n = 8) vs. non-silencing control (shCTRL, n = 4) HT29 cells, and H3K9me2^High (n = 8) vs. H3K9me2^Low (n = 8) HIEC cells at 72 h post-seeding (***: p ≤ 0.0001). F Quantitative PCR assessment of intestinal differentiation markers expression in G9a knockdown, BIX-01294 (1 μM, 48 h) and UNC0642-treated (2.5 μM, 48 h) HT29 cells, as well as BIX-01294 (1 μM, 48 h) and UNC0642-treated (2.5 μM, 48 h) HIEC cells (n ≥ 3, ***: p ≤ 0.0008; **: p ≤ 0.008; *: p ≤ 0.047; #: p = 0.052). Results are expressed as mRNA fold-change vs. respective controls (shCTRL and DMSO). GAPDH was used as a reference gene.