Figure. 3: Purine nucleotide production downstream of serine metabolism constrains cell proliferation when NAD+ regeneration is impaired.

a, Hierarchical clustering and heat map indicating relative metabolite levels measured using LCMS from A549 cells cultured for 48 hours in media with or without serine and with or without 20 nM rotenone as indicated. b, Schematic showing how ¹⁵N label is incorporated from amide-¹⁵N-glutamine into the purines AMP and GMP. Two ¹⁵N labels are incorporated into newly synthesized AMP, while three ¹⁵N labels are incorporated into newly synthesized GMP. c, Total levels, and labeling of ATP and GTP from amide-¹⁵N-glutamine as determined by LCMS in A549 cells cultured in media with serine, or in media lacking serine for the indicated times. d, Levels of the purine synthesis intermediates GAR and AICAR measured using LCMS in A549 cells cultured with or without serine and with or without formate as indicated. GAR and AICAR each precede steps in purine synthesis that involve addition of a 1C unit derived from formate. GAR, glycineamide ribonucleotide; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide. e, Total levels and labeling of ATP and GTP from 5-¹³C-hypoxanthine in A549 cells cultured with or without serine, 20 nM rotenone, and 5-¹³C-hypoxanthine as indicated. Metabolites were analyzed by LCMS. f, Proliferation rates of A549 cells cultured with or without serine, formate, and/or hypoxanthine, and treated with DMSO or 20 nM rotenone as indicated. Data shown are mean (+/− standard deviation) of 3 biological replicates.