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. 2023 Nov 16;21:328. doi: 10.1186/s12964-023-01335-6

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — Effects of candidate genes on glioblastoma cell fitness were validated with functional assays in vitro. A Scheme of validation experiments for novel EpiDoKOL essentiality hits. TD: post-transduction day. B Representative images of long-term clonogenic assay of cells infected with sgRNAs against selected hits and statistical analysis. Quantification of colonies was performed by ImageJ software. C Results of GFP competition flow cytometric assay for selected hits. Day 0 refers to the day of the cell seeding at TD 7. D-F Cell viability (D), Caspase 3/7 activity (E) and AnnexinV analysis (F) conducted on U373 cells upon depletion of ASH2L, RBX1 and SSRP1 genes. G Western blot analysis of ASH2L protein levels upon transduction of U373 cells with g-ASH2L or g-NT. H Western Blot analysis for cleaved Caspase3 and PARP in U373 cells at day 6 and 14 post-transduction with g-NT control or g-ASH2L. I Western Blot analysis for H3K4 mono and trimethylation levels in U373 cells 6 days post-transduction with g-NT control or g-ASH2L. P values determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001