Figure 9. Effects of glucosamine (GlcN) on the expression of β-1,6-GlcNAc-branched N-glycan in the human retinal pigment epithelial cell line (ARPE-19) cells and the regulation of ARPE-19 cell proliferation by N-glycan branching. ARPE-19 cells at 80% confluence were treated with 2 μg/ml tunicamycin, 30 mM GlcNAc, 30 mM glucose, 2.5 mM GlcN, or 5 mM GlcN for 24 h. The cells were harvested, incubated with fluorescein isothiocyanate (FITC)–L-PHA (10 μg/ml), and L-PHA lectin binding was analyzed by flow cytometry. A: Binding of L-PHA lectin to GlcN-treated cells are compared with those in the positive (30 mM GlcNAc or 30 mM glucose) and negative (tunicamycin) control cells. B: The effect of GlcN on L-PHA lectin binding is dose-dependent. Cells were cultured in serum-free medium containing GlcN (2.5 mM or 5.0 mM) or high glucose (Glc, 30 mM) for 24 h. C: The expression of epidermal growth factor receptor (EGFR) in ARPE-19 cells treated with 2.5 mM or 5.0 mM GlcN or 30 mM glucose analyzed by western blot. D: CFSE staining of human donor retinal pigment epithelium (RPE) with various levels of N-glycan branching. The data are representative of at least three independent experiments.