Fig. 1. MYC drives continued metabolic demand after withdrawal of glutamine.

A MRC-5 cells expressing MYC-ER^T2 (MYC) or vector control (VEC) were stimulated for 24 h with 200 nM 4-OHT, and glutamine was then withdrawn for 16 h (GLN−). Apoptosis was assessed by annexin V/PI staining. An ordinary one-way ANOVA was used to determine statistical significance (n = 3 biological replicates, representative of three independent experiments). B Cells were treated with 4-OHT for 24 h, and glutamine was then withdrawn for 12 h. A 30 min pulse with 5-ethynyl uridine (EU) revealed nascent transcripts. EU incorporation was revealed with a fluorescent azide (with DAPI counterstained). C High-content imaging of EU incorporation at the stated times in cells treated as above or with 500 μM actinomycin D. A two-way ANOVA was used to determine statistical significance (n = 3 biological replicates). D Measurement of global translation through ¹³C₆,¹⁵N₂-lysine incorporation and shotgun proteomics. E Cells were switched to DMEM lacking the indicated amino acids and apoptosis was quantified by Incucyte caspase 3/7 analysis. A two-way ANOVA was used to determine statistical significance (n = 3 biological replicates). F LC-MS quantitation of glutathione in cells following 16 h glutamine withdrawal or BSO treatment. A two-way ANOVA was used to determine statistical significance (n = 3 biological replicates). G AnnexinV/PI staining in cells treated as in (F). A two-way ANOVA was used to determine statistical significance (n = 3 biological replicates). For all statistical tests, P ≤ 0.05 was considered significant, and error bars show the standard error of the mean.