Fig 1: Association of glutamine-carbon metabolism with its nitrogen assimilation under hypoxia. a A schematic to show the metabolism of glutamine carbon and nitrogen in the pyrimidine biosynthesis. b Proliferation of HeLa cells with or without knockdown of DHODH, CAD, and GOT1 cultured under hypoxia and normoxia for 3 days. Values are the means ± SEM of triplicate experiments. Western blot to validate the knockdown of DHODH, CAD, and GOT1. c Excretion of dihydroorotate and orotate to medium from cells cultured under hypoxia and normoxia for 8 h. d Relative ammonia excretion from HeLa/shScramble, HeLa/shCAD, and HeLa/shGOT1 cells cultured under hypoxia and normoxia for 8 h. Values are the means ± SEM of triplicate experiments. e, f Relative cellular dihydroorotate and orotate in HeLa/shScramble, HeLa/shCAD, and HeLa/shGOT1 cells cultured under hypoxia and normoxia for 8 h in the presence or absence of 10 mM aspartate. g Mass isotopomer analysis of acetyl-CoA in HeLa/shScramble, HeLa/shCAD, and HeLa/shGOT1 cells cultured with the medium containing 1 mM of 13C5-glutamine under hypoxia and normoxia for 8 h. h, i The relative abundance of cellular dihydroorotate and orotate in HeLa cells cultured under hypoxia and normoxia for 8 h in the presence or absence of 2 mM dimethyl a-ketoglutarate (DMaKG). j Excretion of dihydroorotate to medium from HeLa cells cultured under hypoxia and normoxia for 8 h in the presence or absence of 2 mM DMaKG. k Proliferation of HeLa/shScramble, HeLa/shCAD, and HeLa/shGOT1 cells cultured under hypoxia for 3 days in the presence or absence of 100 µM uridine and/or 2 mM DMaKG. Values are the means ± SEM of triplicate experiments. All cultures were supplied with 10% dialyzed serum. Values are the means ± SEM of three independent experiments, if not specified. *p < 0.05; **p < 0.01 (Student’s t-test)