Fig. 1. Apoptin-induced cell death is independent of FADD and caspase-8. (A) MCF7 and PC-3 cells treated with recombinant TAT-GFP (1 µM) or with recombinant TAT-apoptin (1 µM), and counterstained with DAPI. TAT-GFP localizes both to the nucleus and to the cytoplasm, whereas TAT-apoptin is found almost exclusively in the nuclei (arrows). TAT-apoptin was detected using an anti-haemagglutinin-tag antibody; the tag is incorporated between TAT and the apoptin itself within the fusion protein. Cells treated with TAT-GFP show normal morphology, whereas some of the cells treated with TAT-apoptin show typical apoptotic morphology (cell shrinkage, nuclear condensation). (B) Apoptosis detection in peripheral blood mononuclear cells treated with recombinant TAT-GFP (1 µM) or recombinant TAT-apoptin (1 µM) for the indicated time, or with 5 µg/ml Actinomycin D as a positive control. Some samples were also treated with IL-2 (5 U) in order to counteract the spontaneous apoptosis. (C,D) Measurement of apoptosis in Jurkat caspase-8^(-/-) cells (C), Jurkat cells expressing FADD-DN (D), and control cells treated with TAT-apoptin for the indicated time periods. To exclude solvent- or TAT-peptide related effects, some control cells were treated with the recombinant TAT-GFP (1 µM) instead of TAT-apoptin treatment. (E) Apoptosis detection, measured by flow cytometry, in Jurkat cells either left untreated or treated with the agonistic anti-CD95 antibody (0.1 µg/ml) for 8 hours. Treated Jurkat caspase-8^(-/-) cells and Jurkat cells expressing FADD-DN are also shown. The percentages of apoptotic cells (mean values of three independent experiments) are shown. (F) Jurkat and peripheral blood mononuclear cells (PBLs), isolated by Ficoll-Paque^TM PLUS gradient (Amersham Biosciences) treated with the recombinant TAT-Apoptin (1 µM) for 12 hours. TAT-apoptin was detected by an anti-HA antibody, as in A. Cells are counterstained with DAPI.