Fig. 2. CD95 capping does not correlate with induction of apoptosis. (A) Jurkat and (B) H9 cells were pre-treated for 1 hour with cytD or zIETD and were then incubated with 200 ng/ml CD95 for 2 hours (Jurkat) or 6 hours (H9) to induce apoptosis. Cells were then labeled and analyzed for the presence of activated caspase-3, by flow cytometry. The data are mean values (±s.e.m.) from a minimum of three separate experiments. (C) Nuclear morphology of CytD- and CD95-treated cells was further analyzed by DAPI labeling. Typical apoptotic cells with condensed chromatin can be seen in the CD95 treated samples. CytD treatment did not inhibit the nuclear alterations associated with apoptosis. (D) Cells were treated as in A. and were then assessed for overall cleavage of caspase-8 by immunoblotting. Overall caspase-8 cleavage does not correlate with the capping of CD95 (Fig. 1C,D). Representative immunoblots from two separate experiments are shown.