FIGURE 7.

p62 regulates caspase-8 aggregation and apoptosis by TRAIL alone or combined with ABT-263. A, HCT116 cells containing stable expression of mCherry-p62 were transfected with a VN- or VC-labeled procaspase-8 (C360A) pair (as per Fig. 5) and were treated with TRAIL alone or combined with ABT-263. Localization of mCherry-p62 and caspase-8-Venus were determined by fluorescence confocal microscopy. B, HCT116 and DLD1 cells were treated with TRAIL and ABT-263 for 24 h at a fixed ratio, and cell viability was determined using MTS assay. The CI was calculated, as described under “Experimental Procedures,” and an isobologram was plotted. A CI value <1 indicates a synergistic interaction. C, HCT116 cells with stable expression of p62 versus control (ctrl) shRNA were incubated with TRAIL (10 ng/ml) ±ABT-263 (2 μm) at the indicated doses for 8 h. Expression of caspase cleavage was analyzed by immunoblotting. DMSO, dimethyl sulfoxide.