Treatment with cisplatin causes relocation of ezrin. MCF-7 cells were treated with DMSO or cisplatin (5 μg/ml) for 2 h. (A) Cells were fixed and stained with ezrin-specific polyclonal antibody (green channel) before laser-scanning confocal microscopy. Nuclei were visualized using DRAQ5 nuclear dye (red channel). Shown are representative images of multiple experiments. Representative areas are enlarged in the insets. (B) Cells were homogenized and fractionated into cytosolic and membrane fractions by centrifugation at 100,000 g. Amount of ezrin in total, cytosolic, and membrane fractions (concentrated eightfold) was detected by Western blotting. (C) MCF-7 cells (5 x 10⁵ cells/10-cm plate) were treated with DMSO or cisplatin (5 μg/ml) for the indicated time points up to 2 h. Levels of p-ezrin and total ezrin were detected by specific rabbit polyclonal antibodies. (D) Cells treated with DMSO or cisplatin (5 μg/ml) for 2 h were stained for p-ezrin (green channel) and phalloidin (red channel). Blots from three independent experiments were quantitated using NIH ImageJ (*, P < 0.05). Images are representative of three independent experiments. Bars, 20 μm.