TPA and S-1P inhibit cyt c and Smac/DIABLO release, caspase-3 activation, and loss of viability induced by anti-Fas in Jurkat cells.

(A) Cytosolic extracts from Jurkat cells treated for 3 hours with 50 ng/mL anti-Fas antibody alone or after pretreatment with 50 nM TPA or 5 μM S-1P for 15 minutes were subjected to 15% SDS-PAGE and immunoblotted with anti–cyt c, anti-Smac/DIABLO, and anti-COX II (cytochrome oxidase serves as a marker for mitochondrial contamination of cytosolic extracts). A mitochondrial extract from nontreated cells (mit. fr. Con) was used as a positive control for cytc, Smac/DIABLO, and COX II. (B) Proteins from duplicate extracts were analyzed by immunoblotting with anti–caspase-3 antibody. The mobilities of the 32-kd precursor (p32) and proteolytically processed forms (p20 and p17) are indicated. Similar results were obtained in 3 independent experiments. (C) Jurkat cells were treated for 5 hours in the conditions described above. Loss of viability was assessed by the MTT assay. Samples were made in quadruplicate. Mean values with standard deviation are shown.