Essential role of ROS for caspase-3 activity after TNF-α stimulation. (A) Inhibition of p38 or PI3Ks does not affect caspase-8 or caspase-3 cleavage. Primary human neutrophils were incubated in the presence or absence of the p38 inhibitor SB203580 (1μM) or the PI3K inhibitor wortmannin (100nM) for 1 hour, and then stimulated with TNF-α (50 ng/mL) or anti-FAS antibody. Cells were harvested after 4 hours for analysis of caspase-8 processing (left panel) or 8 hours for analysis of caspase-3 processing (right panel) of culture. A representative immunoblot is shown, and protein expression levels of the active fragment p18 and p17, respectively, were quantified relative to the fresh sample (n = 4). (B) Caspase-3, but not caspase-8, activity is controlled by ROS. Caspase-8 (IETDase) and caspase-3 (DEVDase) activities were assessed by a colorimetric or fluorometric in vitro caspase activity assay. Neutrophils were harvested immediately after isolation (Fresh), left untreated (Control), or incubated with 20μM DPI, 100nM wortmannin, or 1μM SB203580 before stimulation with TNF-α (50 ng/mL). Neutrophils were also stimulated with 1 μg/mL of anti-FAS antibody. Cells were harvested and lysed at 4 hours for analysis of caspase-8 activity or at 8 hours for analysis of caspase-3 activity. Caspase-8 and caspase-3 activities are shown relative to the caspase activity detected in fresh neutrophil lysates (set as 1) (n ≥ 3).