Figure 3.

Glucosamine suppressed the IL-6/JAK/STAT3 signaling pathway in DU145 cells. (A) Glucosamine decreased IL-6 binding to DU145 cells. Cells cultured with or without 2 mM glucosamine for 24 h, and then incubated with a fluorescently labeled IL-6 followed by FACS analysis. Gray histogram represents IL-6 binding to cells without glucosamine treatment, bold line histogram represents IL-6 binding to glucosamine-treated cells and dotted line histogram represents negative control, cells without fluorescently labeled IL-6. (B) Western blot analysis of cells cultured with or without glucosamine following the IL-6 treatment. Cells cultured in serum free medium with or without 2 mM glucosamine for 24 h and then treated with 2 ng/ml IL-6 for 15 min. Whole-cell lysates were subjected to immunoblotting using antibodies specific for gp130, phospho (Tyr1007/1008)-JAK2 (p-JAK2), phospho (Tyr705)-STAT3 (p-STAT3), phospho (Tyr542)-SHP2 (p-SHP2) and actin (loading control). The open arrow indicates N-glycosylated gp130 and the filled arrow indicates N-glycosylation deficient gp130. (C) Tunicamycin decreases IL-6 binding to DU145 cells. Cells were cultured with or without 1 μg/ml tunicamycin for 24 h and then incubated with a fluorescently labeled IL-6 following FACS analysis. Gray histogram represents IL-6 binding to cells without tunicamycin treatment, bold line histogram represents IL-6 binding to tunicamycin-treated cells and dotted line histogram represents negative control, cells without fluorescently labeled IL-6. (D) Western blot analysis of cells cultured with or without tunicamycin followed by the IL-6 treatment. Cells cultured in serum free medium with or without 1 μg/ml tunicamycin for 24 h and then treated with 2 ng/ml IL-6 for 15 min. Whole-cell lysates were analyzed and data were presented in the same way as for glucosamine (Figure 3B). The binding assays and each blot are a representative of three independent experiments.