Fig. 1.

Effects of ATR and dCK inhibition on G1-S transition and substrate utilization for dCTP biosynthesis. a, b Flow cytometry analysis of EdU incorporation in CEM T-ALL cells treated with VE-822 (1 µM) and/or dCKi (DI-82,1 µM) for 6 a and 12 h b following release from G1 arrest, respectively. Bar graphs summarize the percentage of cell populations in S1 (early S-phase) and S2 (mid to late S-phase) at 6 and 12 h (mean ± s.d., n = 2, one-way ANOVA, Bonferroni corrected). Plots are representative of two independent experiments. c Comparison of metabolite labeling by [¹³C₆]glucose in CEM T-ALL cells treated with VE-822 and/or dCKi for 12 h following release from G1 arrest. Number of metabolites exhibiting alterations in [¹³C₆]glucose labeling greater than 15% with significance at a false discovery rate ≤ 20% are indicated. d Percent glucose labeling of ribonucleotides intermediates in the de novo dCTP biosynthesis (mean ± s.d., n = 6, one-way ANOVA, Bonferroni corrected). e Workflow for targeted LC-MS/MS-MRM analysis of dCTP incorporated into newly replicated DNA using a triple quadrupole mass spectrometer. See text for details and Supplementary Fig. 4 for the LC-MS/MS-MRM analysis of dCTP pools. f, g Contributions of the de novo and salvage pathways to dCTP pools f and dCTP incorporated into newly synthesized DNA g in CEM cells treated with VE-822 and/or dCKi after release from G1 arrest (mean ± s.d., n = 3). Results are representative of two independent experiments. NT = Not treated, V = VE-822, D = dCKi, V + D = VE-822 + dCKi. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ATR ataxia telangiectasia and Rad3-related protein, EDU 5’-ethynyl-2’-deoxyuridine, FDR false discovery rate, RNR ribonucleotide reductase, CTPS1/2 CTP synthase 1/2, R5P ribose 5-phosphate, OMP orotidine monophosphate, rCDP cytidine diphosphate, CTP cytidine triphosphate, UTP uridine triphosphate, dCTP deoxycytidine triphosphate, LC-MS/MS-MRM liquid chromatography tandem mass spectrometry operating in multiple reaction monitoring