Effect of Zfra on TRADD, FADD, or RIP-mediated cell death. ME180, HEK-293 and DU145 cells were transfected with cytotoxic doses of TRADD, FADD, or RIP cDNA (200 ng/well), in the presence or absence of EGFP-Zfra (200 ng/well) by CaPO4. These cells were cultured for 48 hr and then stained with crystal violet. In Zfra-sensitive ME180 and HEK-293 cells, Zfra and TRADD increased cell death in an additive manner (n = 8; p < 0.001, TRADD alone versus TRADD/Zfra in combination; Student's t tests), whereas no additive effect was shown for Zfra with FADD or RIP (n = 8; p > 0.05, FADD or RIP alone versus Zfra/FADD or Zfra/RIP; Student's t tests). In Zfra-resistant DU145 cells, Zfra alone did not induce death, but enhanced the death by ectopic TRADD, FADD and RIP by ~100–150% increases (n = 8; p < 0.001; Student's t tests). EGFP-Zfra was expressed in approximately 60–70% of total cells, as visualized by fluorescence microscopy.