Figure 6

Impairment of Smac release requires Smac–XIAP interaction but not effector caspase inhibition. (a) The duration of Smac-YFP release in response to TRAIL/CHX (100 ng/ml, 1 μg/ml) or STS (1 μM) was quantified for XIAP-deficient HCT-116 cells expressing Cerulean, C-XIAP-C or variants thereof bearing W310A or D148A point mutations. Data are means from n=4 control cells, or 8, 11 and 10 cells per group (TRAIL/CHX) or 6, 5 and 7 cells per group (STS). Error bars represent S.E.M. ^*Significantly prolonged Smac release (P<0.05; ANOVA and subsequent Tukey's test; NS, not significant). (b) Comparison of amounts of Smac-YFP released in response to TRAIL/CHX or STS. Cellular fluorescence S.D. for n=4 control cells, or 8, 11 and 10 cells per group (TRAIL/CHX) or 6, 5 and 7 cells per group (STS) were compared at 1, 2 and 3 h after onset of Smac release. Data are shown as means; error bars represent S.E.M. ^*Significant mitochondrial Smac retention (P<0.05; ANOVA and subsequent Tukey's test). (c) Representative images of Smac-YFP fluorescence in HCT-116 XIAP^−/− cells expressing C-XIAP-C or its variants before and 60 min after onset of Smac-YFP release. Full Smac release was only observed in cells expressing C-XIAP-C W310A. Scale bar=10 μm. (d) The duration of Smac-YFP release in response to 100 ng/ml TRAIL/1 μg/ml CHX was quantified for HeLa cells expressing Cerulean, C-XIAP-C or C-XIAP-C W310A. Data are means from n=9, 7 and 7 cells per group. Error bars represent S.E.M. ^*Significantly prolonged Smac release (P<0.05; ANOVA and subsequent Tukey's test; NS, not significant). (e) Comparison of amounts of Smac-YFP released in response to 100 ng/ml TRAIL/1 μg/ml CHX. Cellular fluorescence S.D. for n=9, 7 and 7 cells per group were compared at 1, 2 and 3 h after onset of Smac release. Data are shown as means; error bars represent S.E.M. ^*Significant mitochondrial Smac retention (P<0.05; ANOVA and subsequent Tukey's test)