Fig. 5. Kinetics of TNF and FasL-induced caspase-8 activation. (A) HeLa-TNF-R2 cells were pretreated with CHX (2.5 µg/ml) for 3 hours. Cells were then stimulated for the indicated times with crosslinked FasL (100 ng/ml), TNF (10 ng/ml) alone or in combination with anti-TNF-R2 IgG (2 µg/ml). In an additional group, cells were stimulated with anti-TNF-R2 IgG for 6 hours before TNF treatment. Cell lysates were prepared and procaspase-8 and -3 processing was analyzed by immunoblotting. (B) Cells were treated as described above with crosslinked FasL (), TNF () or TNF and anti-TNF-R2 IgG (costimulation: ; prestimulation: ). After the indicated times, the cells were lysed and analyzed with respect to caspase-8 and caspase-3 activity with fluorogenic substrates. (C) HeLa cells pretreated with CHX (2.5 µg/ml) for 3 hours (left panel) and Kym1 and SKW cells, respectively, (right panel) were challenged with TNF (10 ng/ml) or crosslinked FasL (100 ng/ml). After the indicated times the pan caspase-inhibitor z-VAD-fmk (20 µM) was added and after overnight incubation cell viability was determined by crystal violet staining (HeLa, Kym1) or by the MTT method (SKW).