Fig. 2: GCN2 is playing a pro-apoptotic role in cell death induced by glutamine deprivation.

A HCT116 cells were cultured for 15 h in the presence or absence of glutamine, with or without 1 µM A92. Western blotting was performed to examine eIF2-α phosphorylation, eIF2-α, ATF4, and CHOP levels. Levels of phosphorylated eIF2-α relative to total eIF2-α protein were quantified by ImageQuant TL analysis software (Cytiva, USA). GAPDH was used as protein-loading control. B HCT116 cells were transfected for 48 h with either a Scrambled oligonucleotide (Sc) or two different siRNAs targeting GCN2 (siGCN2#1 and #2). After transfection, cells were incubated for 24 h in complete or glutamine-depleted medium and levels of eIF2α phosphorylation, eIF2α, GCN2, ATF4 and CHOP were assessed by Western blotting. C HCT116 cells were incubated for 24 h in medium with or without glutamine in the presence or absence of 1 µM A92 and apoptosis assessed as described in “Materials and methods” section. D HCT116 cells were transfected for 48 h as described in B. After transfection, cells were incubated for 24 or 48 h in complete or glutamine-depleted medium and apoptosis was then assessed. In C and D, data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; two-way ANOVA test. Tukey’s multiple comparison test.