Fig. 3: Effects of candidate genes on MDA-MB-231 fitness were validated with functional assays in vitro.

A Venn diagram showing cell line specific or common genes that are found in p < 0.05 cutoff. 15 genes in bold show TNBC specific epigenetic modifiers that were depleted in all three TNBC cell lines. Others are the genes that were commonly depleted in two different TNBC cell lines but not in HMLE. B Gene set enrichment analysis with newly curated ‘epigenetic complexes’ gene sets. Normalized enrichment scores demonstrating negative enrichment of epigenetic complexes in MDA-MB-231 cells. C Summary of dual-color competition assay for in vitro validation of candidate epigenetic modifiers. NT1: Non-targeting control, CDC16: positive control. D Results of dual-color competition assay for selected hits in MDA-MB-231 cells. PT: post-transduction day. E Representative images taken with Cytation5 at Day0 and Day16 of competition assay for MDA-MB-231 cells. mCherry+ cells were infected with Non-targeting sgRNA (NT1) as control while eGFP+ cells were infected with sgRNA targeting the gene of interest. Scale bar: 200 µm. F Representative images of long-term clonogenic assay for MDA-MB-231 cells infected with sgRNAs against selected hits. Scale Bar: 10 mm.G Western Blot analysis of MDA-MB-231 cells after transduction with viruses of indicated sgRNAs at post-transduction day 9. H Annexin V & dead cell assay results of selected genes on two different time points and their statistical analysis. I Cell cycle analysis of selected genes on post-transduction day9 and its statistical analysis. P values determined by two-tailed Student’s t-test in comparison to NT1; *P < 0.05, **P < 0.01, ***P < 0.001.