Fig. 6. Glutamine withdrawal-induced apoptosis does not occur due to loss of asparagine.

A Incucyte Caspase 3/7 apoptosis assay 24 h after treatments shown in 4-OHT-treated BJ fibroblasts expressing MYC-ER^T2. Supplemented amino acids are at 4 mM. An ordinary one-way ANOVA was used to determine statistical significance (n = 3 biological replicates). B Peak area of asparagine 6 h after glutamine withdrawal and asparagine supplementation in cells as in (A). C Peak areas of indicated metabolites 6 h after glutamine withdrawal and amino acid supplementation in cells as in (A). D Time-course evaluation of apoptosis by Incucyte caspase 3/7 apoptosis assay in cells as in (A). E Ratio of ATP/AMP peak intensities in cells as in (A). F BJ fibroblasts expressing MYC-ER^T2 (MYC) or control (VEC) after 72 h of siRNA treatment and 24 h of asparagine supplementation. Apoptosis was assessed by annexin V/PI staining. An ordinary one-way ANOVA was used to determine statistical significance (n = 3 biological replicates). For all statistical test, P ≤ 0.05 was considered significant, and error bars show the standard error of the mean.