Chlorpromazine

Human CSFs were seeded at a density of 7.5 × 10⁴ in 6-well culture dish in MEM medium and treated with either 250 nM recombinant human decorin (rhDcn; PeproTech, Rocky Hills, NJ) or 100 ng/ml recombinant human EGF (rhEGF; R&D Systems, Minneapolis, MN, USA) or the combination of Dcn (250 nM) and EGF (100 ng/ml) for 15, 30, or 60 min. The cells were pretreated with AG1478 (Cayman chemicals company 10010244), a specific EGFR kinase inhibitor at 5 μM for 60 min then incubated with EGF or Dcn. hCSF cells were preincubated with 2.5 mM methyl-β-cyclodextrin (Sigma, St. Louis; M7439) or 7.5 μM chlorpromazine (Tokyo Chemical Industry, C281) for 30 min and then treated with EGF or Dcn. At selected time points, cultures were either fixed with fresh 4% paraformaldehyde solution for immunocytochemistry or used for preparing protein lysate with RIPA buffer for western blot analysis. Each experiment was performed in triplicate.