Figure 6

(a) Overexpression of Bcl-2 and expression of a dominant negative caspase-9 partially reduce Ukrain-induced apoptosis. Jurkat Vector cells, Jurkat Bcl-2 cells as well as Jurkat Caspase-9 DN cells were treated for 24 h with medium or 10 μg/ml Ukrain, for 12 h with medium or 25 μM Etoposide and for 48 h with medium or irradiated with 10 Gy 48 h prior to determination of apoptosis. Apoptosis was quantified by fluorescence microscopy upon Hoechst33342-staining by counting cells with apoptotic nuclear morphology. Data show specific apoptosis (apoptosis rates of treated cells minus apoptosis rates of untreated cells) as 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) Overexpression of Bcl-2 partially inhibits Ukrain-induced mitochondrial damage. Jurkat Vector, Jurkat Bcl-2 as well as Jurkat Caspase-9 DN cells were treated for 24 h with medium or 5, 10 and 50 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. (c) Verification of variant Jurkat clones. Expression of Bcl-2 in Jurkat Vector and Bcl-2 overexpressing Jurkat cells (Jurkat Bcl-2) (upper panel) as well as expression of caspase-9 in Jurkat Vector and Jurkat cells expressing a dominant negative caspase-9 (Jurkat Caspase-9 DN) (lower panel) were verified by Western blot analysis of cytotsolic extracts with the respective antibodies. (d) Overexpression of Bcl-2 and expression of a dominant negative caspase-9 only slightly inhibit Ukrain-induced caspase-activation. Jurkat Vector cells, Jurkat Bcl-2 cells as well as Jurkat Caspase-9 DN cells were treated for 0, 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 antibodies against full length and active caspase-3, caspase-8 as well as PARP and cleaved PARP. Data from one representative experiment are shown.