Figure 5.

Effect of a caspase inhibitor on the cell death induced by FKBP-Fas or FKBP-FADD. (A) Effect of Z-VAD-fmk on Fas or FADD-induced proteolysis of nuclear proteins. Jurkat and its transformants expressing FKBP-Fas (clone, JF-1) or FKBP-FADD (clone, JM-94) were preincubated at 37°C for 1 h in the presence or absence of 500 μM Z-VAD-fmk (Z). The cells were left untreated (−), or treated at 37°C for 6 h with 0.5 μg/ml anti–Fas antibody (Fas) or 0.5 μM FK1012 (FK). The crude lysates were prepared by lysing the cells with 2.5% SDS, and analyzed by Western blotting with antibodies against PARP, lamin B1, and α-tubulin. The positions for intact and cleaved PARP, as well as for the intact lamin B1 and α-tubulin, are indicated by arrows on the right. The each lane contains the crude lysates derived from 2.5 × 10⁴ cells. (B) Effect of Z-VAD-fmk on FK1012-induced DNA degradation. After preincubation with 500 μM Z-VAD-fmk, the indicated cells were incubated at 37°C for 6 h in the presence of 0.5 μg/ml anti–Fas antibody (Fas) or 0.5 μM FK1012 (FK). Total DNA was then electrophoresed on 1.5% agarose gel. (C) Effect of Z-VAD-fmk on the cell death induced by FKBP-Fas or FKBP-FADD. The Jurkat cell transformants expressing FKBP-Fas (JF-1 and JF-19) or FKBP-FADD (JM-84 and JM-94) were preincubated at 37°C for 1 h with (hatched bars) or without (closed bars) 500 μM Z-VAD-fmk, and then treated at 37°C for 6 h with 0.5 μM FK1012. The cell viability was determined by the WST-1 assay, as described in Materials and Methods, and is expressed as a percentage of that obtained without FK1012 treatment.