Figure 3: Asparagine is an amino acid exchange factor.

Relative glutamine (a) and asparagine (b) levels in the medium from LPS2 cells, as measured by LC-MS, before and after amino acid (AA) stimulation following pre-loading of the cells with glutamine (Q) and/or asparagine (N). Serum- and amino acid-starved LPS2 cells were pre-loaded with 2 mM glutamine, 2 mM asparagine or 2 mM glutamine and 2 mM asparagine for 60 min prior to stimulation for 30 min with an amino acid mixture (AA medium) lacking glutamine and asparagine. ‘Blank’ indicates measurements from plates lacking cells. ‘No Pre’ indicates measurements from plates of LPS2 cells not pre-loaded with glutamine or asparagine. Relative intracellular glutamine (c) and asparagine (d) levels as measured by LC-MS in glutamine- and/or asparagine-pre-loaded cells before and after amino acid stimulation. (e) Absolute quantification of intracellular glutamine and asparagine in pre-loaded cells prior to amino acid stimulation. (f) Absolute quantification of extracellular glutamine and asparagine following amino acid stimulation. (g) Changes in extracellular amino acid levels during a 24-h incubation with HeLa cells. The 24-h incubation began either 24 h (left panels) or 48 h (right panels) post doxycycline-induced expression of a scrambled shRNA (Scr) or ASNS shRNA. Values are shown as per cent change from amino acid measurements from identical medium incubated on plates lacking cells, with negative bars indicating cellular consumption and positive bars indicating production. For a–f, error bars denote s.d. of the mean (n=3). For g, error bars denote s.e.m. (n=6). P values were calculated by the Student’s t-test: *P<0.05; **P<0.01; ***P<0.001. NS, not significant.