FIGURE 7.

α-MSH inhibits expression of syndecan-2 paralleled with reduced cell migration. A, B16 cells were treated with α-MSH (1.0 μm) for 24 h. Total RNA was extracted, and expression of syndecan-2 was analyzed by reverse transcription-PCR. β-Actin mRNA was used as the loading control. B, B16 cells were treated with either FGF-2 (300 ng/ml) or α-MSH (1.0 μm). After 24 h, total cell lysates were analyzed by slot blotting. C, B16 cells treated with the indicated amount of α-MSH (μm) for 24 h were incubated with anti-syndecan-2 antibody, and the protein expression level was analyzed by flow cytometry. IgG was used as the negative control. D, B16 cells (5 × 10⁴) pretreated with either FGF-2 (300 ng/ml) or α-MSH (0.5 μm or 1.0 μm) for 24 h were allowed to migrate on Transwell plates as described in the legend to Fig. 3. Columns, average of three independent experiments. E, B16 cells were pretreated with α-MSH (1.0 μm) for 24 h. Transwell cell migration or invasion assay was performed as described in Fig. 3. F, B16 cells were treated with either FGF-2 (300 ng/ml) or α-MSH (1.0 μm), and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed. Columns, average of three independent experiments. *, p < 0.01; **, p < 0.05 versus control.