ASMase is upstream of cisplatin-induced ezrin dephosphorylation. (A) MCF-7 cells were transfected with 10 nM of either SCR or ASMase RNAi oligonucleotides. After 48 h, levels of ASMase and β-actin were determined by Western blotting and quantitated by densitometry. (B) Cells were treated with either DMSO or cisplatin (5 μg/ml) for 60 min. Lysates (30 μg) from each treatment group were used to detect levels of phosphorylated ezrin (p-ezrin) and ezrin by Western blotting. Blots from three independent experiments were quantitated using NIH ImageJ (**, P < 0.01; n.s, non significant). (C) Cells plated on 2-cm confocal dishes were transfected with 20 nM of SCR or two ASMase RNAi oligonucleotides (ASMase RNAi [1] and [2]). After 48 h, cells were treated with either DMSO or cisplatin for 60 min. Then cells were fixed and stained using ezrin-specific polyclonal antibody. Images are representative of three independent experiments. Bars, 10 μm.