Figure 6

CXCR1 and CXCR2 activate ERK1/2 phosphorylation and mediated melanoma cell growth and motility. (A) For ERK phosphorylation, A375P cells overexpressing CXCR1 or CXCR2 were stimulated with CXCL-8 (10 ng ml⁻¹) for 0 or 30 min (left and middle panels). Effect of ERK1/2 inhibitor was examined by treating the cells for 1 h and stimulated with CXCL-8 for 30 min (right panel). Cells were lysed, and equal amounts of protein were analysed by western blot analysis using antibodies against pERK1/2 and ERK1/2 antibody. Bound immunocomplexes were detected using ECL Plus chemiluminescence detection reagent. (B) For Growth, A375P-transfected cells were seeded (1000 cells per well) in 96-well plates and incubated with serum-free medium containing CXCL-8 (10 ng ml⁻¹) with or without PD98059 (20 μ M). Cellular growth was determined at 72 h by MTT assay. The values are average absorbance±s.e.m. (C) A375P-transfected cells treated for 1 h with 20 μ M PD98059 were used for migration assay as described in Materials and Methods. Migrated cells were counted in 10 random fields (200 × ). The values are number of migrated cells±s.e.m. This is a representative of three experiments done in triplicate. ^*Significantly different from control cells (P<0.05).