Roles of ASCC2 and ASCC3 in PF8503-dependent toxicty.
(A) Comparison of the Rho phenotype in the CRISPRi screen to relative cell viability in individual gene knockouts. K562_dCas9-KRAB cells with individual sgRNAs targeting genes of interest were competed against cells with a scrambled sgRNA, in the presence of 7.5 μM PF8503. Experiments were carried out in biological triplicate with the average log2(fold change) and standard deviation shown. (B) Effect of treatment with PF8503 (7.5μM) on K562-dCas-KRAB cell lines expressing two different sgRNA targeting either ASCC2 or ASCC3 expression. The apoptotic index, defined as the ratio of Caspase 3/7 levels to ATP levels, measured after 6 days of 7.5 μM PF8503 or DMSO control treatment. Experiment performed in triplicate, with the average and standard deviations shown. (C) Western blot of immunoprecipitation of ASCC3 from the cytoplasm of HEK293T cells. Input, cell lysate; FT, flow-through supernatant; Wash 1 and 4, Bead washes; Elution, Proteins extracted from beads. Gel is representative of duplicate experiments. (D) Western blots obtained after isolation of 80S ribosomes from K562-dCas9-KRAB cell lines expressing scrambled sgRNA, using a sucrose cushion at low (200 mM) or high (400 mM) potassium acetate concentration. Gels are representative of experiments carried out in duplicate.