Metabolic stable isotope tracing

[U-¹³C]aspartate tracer (CLM-1801-H-0.25) were purchased from Cambridge Isotope Labs Inc. Stable isotope tracing experiments to determine isotopologue distributions in soluble metabolites were performed as described previously^63,64. The control and arginine-starved (48 h) MDA-MB-231 cells were incubated in [U-¹³C]aspartate containing medium during the last 6 h of arginine starvation. The intracellular metabolites were harvested, derivatized and measured with gas chromatography mass spectrometry. For simplicity, only the m + 4 isotopologue of metabolites were shown as the products of the first-round aspartate catabolism.

For ¹³C-glucose labeling experiments, cells were cultured in full growth medium or arginine depleted medium for 48 h. [U-¹³C]glucose (10 mM) tracer medium with or without arginine were applied to cells for 6 h prior to harvesting. Isotope-labeled aspartate and glucose tracer medium was prepared from phenol red-free, glucose-free, arginine-free, lysine-free, glutamine-free DMEM (Gibco) supplemented with 10% dialyzed FBS, DMEM-levels of L-lysine and L-glutamine.