Figure 1. Effect of nerve growth factor on cell proliferation and the cell cycle in human corneal epithelial cells. A: Human corneal epithelial cells (HCECs) cultured in vitro displayed a polygonal pattern (left panel). Immunostaining for cytokeratin 12 (green) followed by Hoechst staining (blue; right panel) is shown. B: HCECs were seeded onto 96 well plates at a density of 6×10³ per well in defined keratinocyte serum-free medium (K-SFM), and then treated with recombinant nerve growth factor β-NGF at 5 ng/ml and subjected to a cell proliferation assay for up to 6 days. C: HCECs at passage 1 were plated onto 60-mm dishes. The cell cycle was analyzed by flow cytometry with cells treated identically at day 4 of the cell proliferation assay (upper panel), and the percentage of each phase (G₁-M) is indicated on the right (lower panel). Experiments were performed in triplicate and statistically analyzed with the Student t test. The results are shown as mean±standard deviation (SD). All p-values (*) were considered statistically significant when p<0.05.