Figure 2.

TRAIL and bortezomib cooperate to induce caspase-dependent apoptosis. (a) The 089 and 090 cells were treated with 50 ng/ml TRAIL and 2.5 ng/ml BTZ alone or in combination with the caspase inhibitor zVad (20 μM) and/or the RIP1 kinase inhibitor nec-1 (50 μM). Cell survival was measured 24 h later by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis. Bars represent mean cell viability normalised to untreated control cells and error bars indicate S.E.M. of four independent experiments. P-values were determined by two-way ANOVA (*P<0.05, **P<0.01 and ***P<0.001). (b) Quantification of Annexin V and PI single- or double-positive populations in 089 and 090 cells treated with 50 ng/ml TRAIL and 2.5 ng/ml bortezomib combined with zVad and/or nec-1 as before. Bars represent the percentage of dead cells that were stained positive for Annexin V, PI or both and error bars indicate S.E.M. of three independent experiments. P-values were determined by one-way ANOVA (**P<0.01 and ***P<0.001). (c) Western blot analysis of caspase-3, -8 and -9 processing in 090 cells treated with TRAIL (T, 50 ng/ml) and bortezomib (B, 2.5 ng/ml) alone or in combination (BT) for 16 h. (d) Cytochrome c release was analysed by western blot analysis of cytosolic fractions from 090 cells treated with TRAIL (T, 50 ng/ml) and bortezomib (B, 2.5 ng/ml) alone or in combination (BT) for 20 h