FIG. 8.

Elimination of chemotherapeutic drug-induced apoptosis by overexpression of mutant TRADD proteins. (A) Schematic representation of the TRADD deletion mutants used in this study. The horizontal bars represent the sequence of TRADD, with black regions corresponding to intact sequences and white region indicating the deleted regions. The apoptosis-inducing ability of each construct was reported previously (25). (B) HT1080 cells were transfected with vector pcDNA3 encoding FLAG-tagged WT and mutant tradd cDNAs. After 24 h of transfection, cell lysates were subjected to Western blot analysis with an anti-TRADD antibody or an anti-FLAG antibody. (C) HT1080 cells were transfected with plasmid pcDNA3-EGFP together with the generated pcDNA3 plasmid containing FLAG-tagged tradd cDNAs. After transfection for 24 h, HT1080 cells were treated with 10 μg of VP-16 per ml for an additional 36 h. After fixation, cells were stained with DAPI. Nuclear fragmentation was visualized with a fluorescence microscope. The number of surviving cells was determined by counting 100 EGFP-positive (transfected) cells not showing nuclear condensation and fragmentation. Each vertical bar represents the mean ± the standard deviation of three independent experiments.