Defective apoptotic activities of tumor-derived

CASP10 mutants. (A) Myc-tagged expression constructs of wild-type (WT) CASP10 or tumor-derived CASP10mutants were transfected into 293 cells. Cell lysates were immunoblotted with anti-Myc antibody. (B) The 293 cells were transfected with 1.3 μg wild-type (WT) caspase 10, Stop367, or vector only, together with 0.2 μg of pEGEF. Twenty-four hours after transfection, cell were fixed in 10% methanol for 15 minutes, and stained with 1 μg/mL DAPI for 15 minutes, and the nuclei were examined by fluorescence microscopy. Arrow indicates apoptotic cells with condensed nuclei. Original magnification × 400. (C) The percentage of apoptosis was measured 48 hours later by DAPI staining (mean ± SD; n = 4). The 293 cells were cotransfected with Fas and TRAIL receptor 2 constructs and a 4-fold excess of each mutant caspase 10. Cell death was analyzed as described in panel B.