Estramustine Phosphate Inhibits TGF-β-Induced Mouse Macrophage Migration and Urokinase-Type Plasminogen Activator Production

Figure 2

EP inhibits TGF-β-induced RAW 264.7 cell migration. (a) Cells were cultured until confluence, and a scratch at the centre of monolayer was made. Then, cells were treated as indicated for 24 hours, fixed and stained. 10 μM EP significantly inhibits the closure of the wound induced by TGF-β 5 ng/ml. (b) RAW 264.7 cells were subjected to chemoattractant response to TGF-β 5 ng/ml by using the Boyden chamber-based assay. CFSE stained cells were allowed to migrate across the 8 μm pore membrane for 18 hours. Microphotographs show fixed cells in the bottom of the membrane (green). EP 10 μM significantly reduced TGF-β chemoattractant potency. (c) EP modified tubulin cytoskeleton in the presence of TGF-β. Cells were treated with TGF-β 5 ng/ml in the presence or absence of EP 10 μM for 24 h. Then, cells were subjected to immunofluorescence analysis for tubulin cytoskeleton (red) and nuclei (blue). (d) Neither TGF-β nor EP modified RAW 264.7 cell proliferation, determined by the MTT assay. RU: relative units. Representative results from three independent experiments are shown. Significant difference between treatments by t-test: .

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