Fig. 5: GCN2-mediated activation of the extrinsic apoptotic pathway upon glutamine deprivation in tumor cells.

A Apoptosis was assessed in pBabe or dnFADD HCT116 cells cultured in the presence or absence of glutamine for 30 h. Endogenous FADD and dnFADD expression were assessed by Western-blotting. GAPDH was used as protein-loading control. B HCT116 (upper panel) or MDA-MB468 (lower panel) cells stably expressing a Scrambled or a caspase-8 targeting shRNA, were incubated in the presence or absence of glutamine and apoptosis was measured at the indicated times (HCT116) or 48 h (MDA-MB468). C HCT116 WT or Bax/Bak KO cells were cultured in the presence or absence of glutamine for 30 h. Following these treatments, procaspase-8 (proC8) and cleaved caspase-8 (cC8) were assessed by Western blotting. Data are presented as mean ± SD from at least three independent experiments. ***P < 0.001; ****P < 0. 0001; two-way ANOVA test. Tukey’s multiple comparison test. D HCT116 cells were incubated in medium with or without glutamine for the indicated times, in the presence or absence of 1 µM A92. Caspase-8 (upper panel) and caspase-3 (cC3) (lower panel) activation was assessed by Western blotting. E HCT116 cells stably expressing a scrambled oligonucleotide (shSc) or a GCN2 targeting shRNA (shGCN2) were cultured in the presence or absence of glutamine for the indicated times. Following these incubations, caspase-8 (cC8, left panel) and caspase-3 (cC3, right panel) activation, and GCN2 levels (right panel) were assessed by Western blotting.