Extended Data Fig. 2: Nutrient-deprived PDA use uridine to support metabolism.

a. Relative RMA upon uridine supplementation with or without glucose and glutamine. n = 4 biologically independent samples per group per cell line. b. Differential changed (P < 0.05) intracellular and c) extracellular metabolites from PATU8988S cells supplemented with 1 mM uridine in glucose-free medium for 24 h, as determined by LC-MS metabolomics. n = 3 biologically independent samples per group. d. Differentially changed (P < 0.05) intracellular and e) extracellular metabolites from DANG cells supplemented with 1 mM uridine in glucose-free medium for 24 h, as determined by LC-MS metabolomics. n = 3 biologically independent samples per group. f. Intracellular and g) extracellular uridine and uracil from DANG cells supplemented with 1 mM uridine in glucose-free medium for 24 h, as determined by LC-MS. Plots in f-g are from the same experiment as d-e. n = 3 biologically independent samples per group. Statistical significance was measured using two-tailed unpaired t-test. Intracellular – comparison between no uridine and 1 mM uridine: *** P = 0.0001 for uridine, ** P = 0.0011 for uracil. Extracellular – comparison between no uridine and 1 mM uridine: *** P = 0.0002 for uridine, ** P = 0.0044 for uracil. h. Mass isotopologue distribution of ¹³C₅-uridine ribose-derived carbon in the displayed metabolites after 24 h culture in a glucose-free medium supplemented with 1 mM uridine. n = 3 biologically independent samples per cell line. Tracing experiments were performed twice in these cells with similar results. Data (a, f, g, h) are shown as mean ± s.d. ADP, adenosine diphosphate; AMP, adenosine monophosphate; GSSG, oxidized glutathione; NADH, nicotinamide adenine dinucleotide; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; X5P, xylulose 5-phosphate. Statistics and Reproducibility: a, n = 4 biologically independent samples per group per cell line. Statistical significance was measured using one-way ANOVA with Tukey’s multiple comparisons test. PATU8988S (comparison between cells cultured in (−) glucose/glutamine/uridine and (−) glucose/glutamine/+uridine: *** P = 0.0007, comparison between (−) and (+) uridine in the presence of glutamine and without glucose: **** P < 0.00001, comparison between (−) and (+) uridine in the presence of glutamine and glucose: P = ns (0.8856)). DANG (comparison between cells cultured in (−) glucose/glutamine/uridine and (−) glucose/glutamine/+uridine: * P = 0.0165, comparison between (−) and (+) uridine in the presence of glutamine and without glucose: **** P < 0.0001, comparison between (−) and (+) uridine in the presence of glutamine and glucose: P = ns (0.7971)). ASPC1 (comparison between cells cultured in (−) glucose/glutamine/uridine and (−) glucose/glutamine/+uridine: **** P < 0.0001, comparison between (−) and (+) uridine in the presence of glutamine and without glucose: **** P < 0.00001, comparison between (−) and (+) uridine in the presence of glutamine and glucose: P = ns (0.9968)).