Fig. 3. TNFR1- and TNFR2-mediated apoptosis in PC60 R1R2 cells are both inhibited by the cowpox caspase inhibitor CrmA. (A) PC60 R1R2 cells were transfected with a combination of the plasmids pCAGGSCrmA and pNFconluc. After selection and subcloning, cell lines were isolated that expressed the CrmA protein and produced elevated levels of luciferase activity after TNF stimulation (named PC60 R1R2 CrmALuc). Control cells were obtained by only transfecting pNFconluc and subsequent screening for luciferase inducibility (PC60 R1R2 luc). Untreated cells were lysed and 100 µg of protein was subjected to CrmA immunoblot. The arrowhead shows the position of the CrmA protein. (B) PC60 R1R2luc and PC60 R1R2CrmAluc cells were cultured in the absence or presence of hTNF (100 ng/ml) or receptor specific muteins (500 ng/ml) for 20 hours and the percentage of propidium iodide-positive cells was determined.