Fig. 1
Generation of ARID1A-knockout (KO) HCT116 cells and screening of epigenetic drug library for synthetic lethality. a Illustration for sgRNA target sites on ARID1A exon 2 for HDR donor plasmid insertion and primers designed for Sanger sequencing. b PCR amplification of ARID1A exon 2 using the designed primers in HCT116 ARID1A+/+ and three ARID1A−/− clones. ARID1A−/− clones #1 and #2 are homozygous ARID1A-KO with HDR donor plasmid insertion into ARID1A gene. Clone #3 is a heterozygous ARID1A−/− containing a HDR insertion mutation and an NHEJ Indel mutation. c Immunoblot analysis showing loss of ARID1A expression in the three ARID1A−/− clones. d Schematic illustration of the synthetic lethality epigenetic drug screening. HCT116 ARID1A+/+ and ARID1A−/− #1 cell lines were screened in parallel with 128 epigenetic drug library in an 8-dose titration format. After incubation with the drug library for 72 h, cell viability was determined by AlamarBlue assay. e A log10-IC50 plot of the screening results. A log10 scale of IC50 values of the drugs against HCT116 ARID1A+/+ and ARID1A−/− cells was plotted. Drugs with selectivity index (SI) > 2 for ARID1A−/− cells were selected and marked as synthetic lethality candidates. f Dose–response curves of HCT116 ARID1A+/+ and three ARID1A−/− clones treated with Aurora A inhibitor I (AURKAi) for 72 h are shown. Error bars represent s.d. (n = 9) from three independent experiments. Survival curve of all three KO clones versus wild-type cells P value < 0.0001, ANOVA. g–k Dose–response curves of HCT116 ARID1A isogenic cell pair treated with AURKAi (g) and known synthetic lethality compounds for ARID1A, including tubastatin A (HDAC6 inhibitor) (h), VE-821 (ATR inhibitor) (i), olaparib (PARP inhibitor) (j), and EPZ-6438 (EZH2 inhibitor) (k), are shown. HCT116 ARID1A+/+ and ARID1A−/− #1 clone were incubated with indicated compounds for 72 h and the cell viability was determined by AlamarBlue assay. Error bars represent s.d. (n = 9) from three independent experiments. ANOVA P value of <0.0001 for AURKAi, tubastatin A, VE-821, and olaparib. ANOVA P value of 0.1629 for EPZ-6438
