Figure 4. zASPG inhibits protein translation and cell proliferation under glutamine depletion only when exogenous asparagine level is low.

A. T47D cells transduced with constitutively expressed empty vector (Ctrl) or zebrafish asparaginase (zASPG) were cultured in various concentrations of asparagine in the absence of glutamine. Cell proliferation was recorded as a cell number fold change 5 days post medium switch. Cells cultured in 2 mM glutamine without asparagine were used as a control. Red box: concentration of asparagine similar to the levels in human plasma. B. LC-MS quantification of intracellular asparagine under conditions as indicated for 16 hours. Relative metabolite levels are shown. Data in A and B are presented as the mean ± SD of triplicates from a representative experiment. C. Ctrl- or zASPG-transduced cells were cultured in conditions as indicated for 24 hours. Puromycin was added at 90 μM for 10 minutes before protein harvest. Whole cell lysates were subjected to Western blotting with puromycin antibody. D. Ctrl- or zASPG-transduced cells were cultured in conditions as indicated for 24 hours. MG-132 (10 μM) was added for 6 hours before harvesting for Western blot analysis. E. Ctrl- or zASPG-transduced MDA-MB-468 cells were inoculated subcutaneously into both flanks of athymic female nude mice. Tumor volumes were recorded weekly. F. Tumor tissues were collected at the end point of the experiment in E. Asparagine and glutamine levels were quantified by LC-MS. The results were normalized to the weight of tumor tissues, and then to the median of all amino acids in each tumor sample. P values for E and F were determined by two-way ANOVA analysis Data in E and F are presented as the mean ± SD of n = 8 from a representative experiment. See also Figure S4.