Activation of ASMase after treatment with cisplatin. (A) MCF-7 cells were treated with either DMSO or cisplatin (5 μg/ml) for 15, 30, 60, or 180 min. Total lysates (100 μg/sample) were subjected to SMase assays (ASMase, NSMase; neutral sphingomyelinase and S-SMase; secretory sphingomyelinase) as described under Materials and methods. Results shown are averaged from three enzymatic assays ± S.E. (*, P < 0.05). (B and C) Cells plated on 2-cm confocal dishes (5 × 105 cells/plate) were transfected with 1 μg of the V5-ASMase plasmid. After 24 h, cells were incubated with either DMSO or cisplatin (5 μg/ml) for 30 min. Localization of ASMase was detected using a V5 monoclonal antibody (green channel) in permeabilized (B) or non-permeabilized cells (C). Sphingomyelin was visualized indirectly using the SM-binding protein, lysenin (red channel). Immunofluorescence was performed using a polyclonal lysenin antibody. Nuclei (NUC) were labeled using DRAQ5 nuclear dye. Shown are representative images of three independent experiments. Bars, 5 μm.
