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).
