Fig. 4. Culture medium defines cell metabolic landscape by altering nutrient exchange rates and metabolic pathways.

(A) Consumption and secretion rates of amino acids and intermediates of glycolysis and the urea cycle in cells cultured in DMEM-F12 and Plasmax. Means ± SEM; n = 3 independent experiments. (B) Correlation analysis between the differential availability of neutral proteinogenic amino acids in Plasmax compared to DMEM-F12 and their respective consumption rates. Means ± SEM; n = 3 independent experiments. P values refer to a two-tailed Pearson test. (C) Consumption of the amino acid bound nitrogen. Means ± SEM; n = 3 independent experiments. (D to I and K) Intracellular abundance of metabolites and metabolite ratios in cells cultured in Plasmax and DMEM-F12. Means ± SEM; n = 3 independent experiments. (J) Western blot showing ASS1 levels in cells cultured in DMEM-F12. Images are representative of three independent experiments. (L) Schematic representation of the urea cycle and expected labeling of argininosuccinate from ¹³C₆ arginine upon reversed ASL activity. (M to O) Intracellular levels of ¹³C argininosuccinate in CAL-120 (M), BT549 (N), and MDA-MB-468 (O) cells cultured for 48 hours in Plasmax and DMEM supplemented with ¹³C₆ and ¹³C₀ arginine, as indicated. Means ± SEM; n = 3 (M) or n = 2 (N and O) independent experiments. P values refer to a two-tailed t test for paired homoscedastic samples.