Figure 2

(a) Ukrain induces morphological alterations indicative for apoptosis. Mock-transfected Jurkat control cells (Jurkat Vector) were treated for 24 h with medium or 10 μg/ml Ukrain and subsequently stained with Hoechst33342. Apoptotic cells show characteristic chromatin condensation and nuclear fragmentation. Micrographs of a representative experiment are shown. (b) Ukrain induces apoptosis in a concentration-dependent manner. Jurkat Vector cells were treated for 24 h with medium or 5, 10 and 50 μg/ml Ukrain. Apoptosis was then quantified by flow cytometric analysis using light scatter characteristics (white bars), determination of the mitochondrial membrane potential Δψm upon staining with the potential sensitive dye TMRE (grey bars) and detection of caspase-activation upon staining with CaspACE^® (black bars). Data show specific apoptosis (% apoptosis in Ukrain-treated cells minus % apoptosis in untreated control cells) as means ± SD (n ≥ 3). (c) Ukrain induces apoptosis in a time-dependent manner. Jurkat Vector cells were treated with medium or 10 μg/ml Ukrain for 3, 6, 12 and 24 h and subsequently analysed by flow cytometry using light scatter characteristics (white bars), determination of the mitochondrial membrane potential upon staining with the potential sensitive dye TMRE (grey bars) and detection of caspase-activation upon staining with CaspACE^® (black bars). Data represent specific apoptosis (means ± SD, n ≥ 3). (d) Ukrain induces activation of caspases. Jurkat Vector cells were treated with medium or 10 μg/ml Ukrain for 6, 12 and 24 h. Caspase activation was determined by Western blot analysis of cytosolic extracts with specific antibodies against caspase-8 and caspase-3 and their respective cleavage products. In addition, caspase-3 activation was verified by detection of the cleavage of the caspase-3 substrate PARP. Data from one representative experiment are shown.