Fig. 7: GCN2-dependent activation of the extrinsic pathway of apoptosis in tumor cells treated with AOA.

A Apoptosis in HCT116 cells cultured for 48 h in complete medium with or without AOA in the presence or absence of non-essential amino acids (NEAA). B, C HCT116 cells were transfected either with a scrambled oligonucleotide (Sc) or with two different siRNAs targeting GCN2 (siGCN2#1 or #2). 48 hours after transfection, cells were either incubated for 16 h in the presence of absence of AOA to assess phosphorylated eIF2α, eIF2α and CHOP levels by Western blotting (B), or during 24 h to measure apoptosis (C). D HCT116 cells were transfected and treated as described in B. TRAIL-R2 and GCN2 levels were assessed by Western blotting. GAPDH was used as protein-loading control. E HCT116 cells stably expressing scrambled, caspase-8, or TRAIL-R2 targeting shRNA, were cultured in the presence or absence of AOA, and apoptosis was measured at 24 or 48 h. Caspase 8 and TRAIL-R2 knockdown were determined by Western blotting. In A, C and E, data are presented as mean ± SD from at least three independent experiments. *P < 0.05; ***P < 0.001; ****P < 0.0001; two-way ANOVA test. Tukey’s multiple comparison test.