Extended Data Fig. 3. CRISPR–Cas9 screen validation.
(a) Differential sensitivity to small molecule inhibitors of the PPP (OT: oxythiamine) or de novo pyrimidine synthesis (brequinar) in glucose vs uridine in UPP1-FLAG expressing K562 (n = 3, P < 2.2 × 10−3, P < 4.9 × 10−6) after 4 days, reported as fold of DMSO. Data are shown as ±SEM with two-sided t-test relative DMSO. (b) Immunoblot analysis of proteins from upper glycolysis, the PPP and pyrimidine salvage in UPP1-expressing K562 cells treated with their corresponding sgRNAs. UCK1 is expressed at low levels in K562 cells and its protein could not be detected. Representative of n = 2 experiments. (c) Simplified representation of the uridine salvage pathway and thymidine synthesis. TUBB and Actin loading controls were performed on the same gels. All growth assays and screens included 4mM L-glutamine and 10% dialyzed FBS.