Drug-induced apoptosis and activation of caspase-8 is independent of FADD signaling. (A and B) HeLa cells (4 × 10⁴) stably expressing a GFP-tagged dominant-negative mutant of FADD (GFP_FADD-DN) or the GFP-tagged vector alone (GFP_vector) were treated with anti-CD95 (1 μg/mL), staurosporine (2.5 μmol/L), etoposide (25 μg/mL), mitomycin C (25 μg/mL) or left untreated. (A) After 24 hours, HeLa-FADD-DN cells were analyzed for the induction of apoptosis under a fluorescent microscope. (B) Subsequently, HeLa-FADD-DN and HeLa-vector cells were lysed in a hypotonic buffer, and the number of hypodiploid apoptotic nuclei was determined in a flow cytometer. Mean values of ± SD from triplicate experiments are shown. (C) HeLa-FADD-DN (1.5 × 10⁶) or HeLa-vector cells were incubated with medium, anti-CD95 (1 μg/mL), staurosporine (2.5 μmol/L) or mitomycin C (25 μg/mL) for the indicated time. Cellular proteins were immunoblotted with anti-caspase-8 as described in Fig 5A. The intermediate cleavage forms (p43 and p41) of caspase-8a and caspase-8b are shown.