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