Fig. 2. Both TNFR1- and TNFR2-mediated apoptosis in PC60 R1R2 cells are blocked by the caspase inhibitor zVAD-fmk. (A) PC60 R1R2luc cells were seeded in microtiter plates and left untreated or were stimulated with saturating amounts of hTNF (100 ng/ml) in the presence (100 µM) or absence of the synthetic peptide caspase inhibitors zVAD-fmk, zDEVD-fmk, zYVAD-cmk, zIETD-fmk or the granzyme B inhibitor zAAD-fmk. Cells were cultured for 20 hours before cell death was measured by cytofluorometric analysis of PI uptake. (B) The experimental procedure was the same as in A. Cells were incubated with hTNF or its receptor-specific muteins R32WS86T or D143NA145R (500 ng/ml) in the absence or presence of zVAD-fmk. (C) Cells were treated for 7 hours in the presence of 100 ng/ml hTNF, in the presence or absence of peptide inhibitors zDEVD-fmk or zVAD-fmk. Cell lysates were analyzed by means of western blotting with a caspase-3 antibody. Pro is the unprocessed procaspase-3, and p20 is the processed form with the black arrowhead showing the normal p20 subunit and the white arrowhead showing a shifted band indicative of a covalent bond between the p20 and zDEVD-fmk. (D) The experimental procedure was the same as in A. Cells were lysed 6 hours after stimulation and luciferase activity determined.