Figure 3

Changes in signalling pathways after HGF. (A) Total cell extracts from starved (st) and HGF-treated cells were immunoprecipitated using anti-Met antibody, and immunoblotted with anti-Met and anti-phosphotyrosine (p-Tyr) antibodies. The experiments were repeated three times, with similar results. (B) MCF-7 and MDA-MB231 cells were exposed to HGF for various times, and western blots of total proteins were performed in triplicate. Representative western blots are shown. Densitometric analysis was carried out for each cell line, ERK1/2, Akt and c-Src protein levels were not changed by HGF, and were taken as 1. The graphs show phosphoERK1/2/ERK1/2 (pERK1/2), phosphoAkt/Akt (pAkt) and phospho-c-Src/c-Src (pSrc) ratios in starved (○) and HGF-treated (•) MCF-7 cells and in starved (▵) and HGF-treated (▴) MDA-MB231 cells. The data are the means±s.e. of experiments repeated three times. ^*P<0.05, ^**P<0.005 and ^***P<0.001 vs respective values for st cells. (C) MCF-7 and MDA-MB231 cells pretreated with HGF for 24 h, with or without 2 μ M c-Src or 8 μ M Akt inhibitor, were used for the Matrigel invasion assay, with CXCL12 in the bottom chamber. We estimated invasion by counting the invading cells on the lower side of the membrane after staining ( × 200 magnification of selected fields). The experiments were repeated three times. The numbers at the bottom indicate the means of migrated cells of 10 fields±s.e. ^*P<0.05 and ^***P<0.001 vs st cells. Western blot analysis of CXCR4 protein levels in 24 h HGF-treated cells, with or without c-Src or Akt inhibitor. The numbers at the bottom indicate the means±s.e. of three experiments. ^*P<0.05 and ^***P<0.001 vs st cells. Cells transfected with NF-κBLuc were exposed to HGF for 24 h, with or without c-Src or Akt inhibitor. The histograms indicate the absolute values for ratios of Firefly/Renilla luciferase activity. The data are the means±s.e. of five independent experiments performed in triplicate. ^*P<0.05 and ^***P<0.001 vs control value; °P<0.05 and °°°P<0.001 vs HGF-treated cells.