Fig. 3.

The BR combination triggers Myc- and GCN2-dependent apoptosis. Indicated cells were treated with vehicle (Un), or the Bortezomib (B), and Everolimus (R) combination (BR, 5 nM and 10 µM). a Western blotting of indicated proteins at 0, 2, 4 and 24 h. ND, not detected. b Western blotting of indicated proteins at 0, 1, 2 and 4 h. (c). GSEA of differential genes in the indicated pair (C2 dataset). The indicated gene set is shown with NES (normalized enrichment score) and corresponding p-value. d Cells were transfected with either scramble or GCN2 siRNA for 24 h, replated for 24 h and treated with BR for 48 h. Attached cells were visualized by crystal violet staining. e Quantification of Annexin V + cells. f Western blotting of indicated proteins, and (g) qRT-PCR analysis of indicated genes at 24 h. The arrow indicates the specific lower band. h Cells were transfected with either scrambled (Ctrl) or MYC siRNA and treated with BR as in d, analyzed by western blotting at 24 h and i apoptosis at 48 h. g, i, values are mean + s.d. (n = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student’s t-test, two tailed). Control (Ctrl) vs. siRNA