PKCδ is upstream of ASMase activation by cisplatin. (A) MCF-7 cells (5 × 10⁵ cells/plate) were treated with DMSO or cisplatin (5 μg/ml) for 5, 30, 60, or 180 min. PKCδ was immunoprecipitated from lysates of each treatment group and subjected to in vitro kinase assay as described in Materials and methods.³²P-MBP was visualized by autoradiography. Average densitometric analysis + S.E. of three independent experiments are plotted. (B) Cells were infected with control LacZ or DN-PKCδ adenoviruses (multiplicity of infection of 50). After 24 h, cells were treated with DMSO or cisplatin for 30 min. Samples of total lysates (100 μg) were then subjected to in vitro ASMase assay. (C and D) Cells overexpressing ASMase (or ASMase^S508A) were infected with LacZ or DN-PKCδ adenoviruses as in B before treatment with either DMSO or cisplatin for 30 min. Localization of ASMase was observed by confocal microscopy using a V5 monoclonal antibody staining in permeabilized cells. Percentage of cells showing plasma membrane translocation of ASMase was quantitated from at least 10 random visual fields each containing 15–20 cells per field. (*, P < 0.05). ns, not significant.