Figure 6.

Exogenous ceramide mimics cytoskeletal effects of cisplatin. MCF-7 cells were treated with D-e-C₁₆-ceramide (5 μM) or vehicle (2% dodecane/98% ethanol) for 2 h. (A) Cells were fixed and stained with ezrin-specific polyclonal antibody (green channel). Nuclei were visualized using DRAQ5 nuclear dye. (B) Cells were homogenized and fractionated into a 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) Cells were treated with D-e-C₁₆-ceramide (5 μM), dh-C₁₆-ceramide (5 μM), or vehicle (2% dodecane/98% ethanol) up to 3 h. Levels of p-ezrin and total ezrin were detected by Western blotting. (D) Cells treated with bSMase (100 mU/ml) for 0, 15, 30, and 60 min were subjected to Western blot analysis for ezrin and p-ezrin levels. (E) Subcellular distribution of ezrin in control cells and cells treated with bSMase (100 mU/ml) for 60 min. (F) Cells treated as in D were stained for p-ezrin (green channel) and phalloidin (red channel). Results are representative of three independent experiments. Blots from three independent experiments were quantitated using NIH ImageJ (*, P < 0.05). Bars, 20 μm.