10.1371/journal.pgen.1008057.g003 Nadège Liaud Nadège Liaud Max A. Horlbeck Max A. Horlbeck Luke A. Gilbert Luke A. Gilbert Ketrin Gjoni Ketrin Gjoni Jonathan S. Weissman Jonathan S. Weissman Jamie H. D. Cate Jamie H. D. Cate Roles of ASCC2 and ASCC3 in PF8503-dependent toxicty. Public Library of Science 2019 compound ubiquitin binding protein ASCC 2 translation quality control proteins Pelota PCSK 9 translational inhibitor LDL genome-wide CRISPRi screen HBS 1L PF 8503 treatment ribosome quality control pathways ASCC 3 acts PELO stall protein synthesis 2019-03-15 17:39:07 Figure https://plos.figshare.com/articles/figure/Roles_of_ASCC2_and_ASCC3_in_PF8503-dependent_toxicty_/7853099 <p>(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 log<sub>2</sub>(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.</p>