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Fig. 6. CD95 capping but not apoptosis is ROCK dependent. (A) Jurkat and (B) H9 cells pre-treated for 1 hour with the ROCK inhibitor Y-27632 were labeled for CD95 alone (first panel; green) or together with F-actin (second panel; red) as in Fig. 1 and Fig. 4B,C. The cells that were pre-treated with Y-27632 did not show any capping of CD95. Maximum projections (left panels) or single overlay z-sections (right panels) from confocal images are shown. Inhibition of ROCK abolishes CD95 capping in both cell types. (C) Jurkat and H9 cells were pre-treated as in A and were then incubated with 200 ng/ml CD95 for 2 hours (Jurkat) or 4 hours (H9) to induce apoptosis. Cells were then analyzed for activation of caspase-3 as in Fig. 2A,B. Inhibition of ROCK did not inhibit CD95-mediated apoptosis. The data represent mean values (mean±s.e.m.) from a minimum of three separate experiments. (D) The nuclear morphology of samples from B were analyzed by DAPI labeling to confirm that the cells were apoptotic. Y-27632 did not inhibit nuclear alterations associated with apoptosis.

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