Figure 6: Functional analyses of F122A and Y8G mutant procaspase 8.

(a) Western blot analysis of DR5 DISC IP carried out in HCT116 cells expressing wild-type and mutant Flag-procaspase 8. Cells were transfected for 24 h prior to treatment. The DR5 DISC was captured 1 h after addition of anti-DR5-conjugated beads to cells. Co-immunoprecipitation of Flag-tagged procaspase 8 and FADD was assessed and pull down of DR5 confirmed. (b) Assessment of caspase 3/7-like DEVD-ase activity in parental and caspase 8 null Jurkat cells treated with 50 ng ml⁻¹ TRAIL for 6 h. The caspase 8 null cells were transfected with empty vector (EV) and wild-type (WT), F122A and Y8G mutant procaspase 8 as indicated. Mean values and s.d. are shown (n=3); *denotes P<0.05 compared with EV control as determined by t-test. (c) Assessment of apoptosis induced by rTRAIL in HCT116 cells overexpressing wild-type and mutant Flag-procaspase 8. Endogenous procaspase was downregulated by transfection with 20 nM of a 3′ UTR-targeted siRNA. Twenty-four hours following siRNA transfection, cells were transiently transfected with each construct for a further 24 h prior to treatment with 5 ng ml⁻¹ rTRAIL for 12 h. Apoptosis was assessed by PARP cleavage. (d) Western blot analysis of FADD and caspase 8 expression in HCT116 cells treated with 20 ng ml⁻¹ TRAIL for 3 h. Following treatment, cell lysates were prepared and passed through a size exclusion column. Fractions of different MW were collected as indicated by trace obtained from known MW standards. The results indicate that p53/55-procaspase 8 is processed to p41/43-caspase 8 in high-MW fractions following TRAIL treatment, with p18-caspase 8 appearing in the lower MW fractions. FADD was present in high-MW fractions in untreated cells, and its presence in the high MW fractions increased following TRAIL treatment. (e) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag-tagged wild-type (WT) or F122A mutant procaspase 8 were treated with 20 ng ml⁻¹ rTRAIL for 3 h prior to cell lysis and separation of low and high MW complexes using a size exclusion column. The expression of Flag-tagged procaspase 8 proteins and endogenous FADD were assessed by western blotting. Only the high-MW fractions are presented.