Effect of anti-HGF neutralizing antibody on PC-3 cell functions A, Serum-starved DU145 cell colonies and scratched monolayers were incubated with CM (serum-free) from 106 PC-3 cells or HGF (5 ng/ml) for 24 h, for the assessment of cell scattering and migration, respectively. Magnification, ×5. Data are from 1 of 3 independent experiments. B, serum-starved DU145 cells were incubated with CM (from 106 PC-3 cells) in the absence (0%, lane 2) or presence of FBS (10%, lane 7), or pure exogenous HGF in a fresh FBS-free medium (5 ng/ml, lane 5) for 15 min, with untreated controls in a fresh medium with 0% (lane 1) or 10% FBS (lane 6) accordingly. In the CM with 0% FBS (lanes 2–4), DU145 cells were pre-treated with anti-HGF neutralizing antibody (anti-HGF, lane 4) or normal mouse IgG1 control (nIgG, lane 3) in a fresh serum-free medium at 37°C for 2 h, to confirm whether c-Met activation if could be triggered by the CM can be blocked by the neutralizing antibody. c-Met signaling molecules were analyzed by Western blot, with total c-Met as a loading control.