Figure 4

(a) Expression of caspase-8 and FADD are not essential for Ukrain-induced apoptosis. Jurkat A3 control cells as well as FADD-deficient and caspase-8-deficient Jurkat cells were treated for 24 h with medium or 5, 10 and 50 μg/ml Ukrain. Apoptosis was then determined by flow cytometry using light scatter characteristics (left panel). Alternatively, A3, FADD-deficient and caspase-8-deficient Jurkat cells were treated for 3, 6, 12 and 24 h with medium or 10 μg/ml Ukrain. Apoptosis induction was then quantified by determination of the mitochondrial membrane potential (Δψm) (right panel). Data show induction of specific apoptosis (means ± SD; n ≥ 3). One-way ANOVA was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, http://www.graphpad.com. (b) Expression of caspase-8 and FADD are not essential for Ukrain-induced caspase-activation. Jurkat A3 as well as FADD-deficient and caspase-8-deficient Jurkat cells were treated for 6, 12 and 24 h with medium or 10 μg/ml Ukrain. Caspase activation was then determined by Western blot analysis of cytosolic extracts with specific antibodies against caspase-3, cleaved caspase-3, PARP and cleaved PARP, respectively. In addition, cells were treated for 3, 6, 12 and 24 h with medium or 10 μg/ml Ukrain and Western blot analysis with specific antibodies against caspase-8 was performed. Data from one representative experiment are shown.