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>