Fig. 6. p145^met is required for survival of serum-starved 5637 cells: effect of HGF-induced down-regulation of p145^met. (A) Normally grown 5637 cells (C, control) were treated with 10% FCS plus 125 ng/ml HGF for 24 hours (H, HGF-treated). After treatment, the cells were further treated for the indicated times (1-24 hours) in new medium containing 10% FCS plus 125 ng/ml HGF (10% FCS) or 125 ng/ml HGF alone (no FCS). After treatment, whole cell extracts were prepared and analyzed by immunoprecipitation and/or immunoblotting as in Fig. 1B, Fig. 4C and Fig. 5B. The positions of pp145^met, p145^met, pp60^src, p60^src, pp44 and pp42 are indicated. (B) Carcinoma 5637 cells (1x10⁶ cells/dish) were cultured in normal conditions (10% FCS) for 24 hours, and then cultured for 24 hours in 10% FCS plus 125 ng/ml HGF () or 10% FCS plus 125 ng/ml HGF plus 1.25 mM CuCl₂ (). After treatment, cells were treated for an additional 48 hours in serum-free media. Cell number was determined at 24, 48, 72 and 96 hours post initial treatment as in Fig. 1A. As a control, data obtained with normally grown cells (10% FCS, 48 hours) and serum-starved cells (– FCS, additional 48 hours) are shown (). (C) Carcinoma 5637 cells were treated as in panel B. After treatment (96 hours post treatment), cell death (black bars) and caspase 3/7 protease activity (grey bars) of the whole cell extracts (20 µg/assay) were determined as in Fig. 1D. Data shown are representative of three independent experiments. Results are the mean ± s.d. of three independent experiments. *P<0.01 compared with levels in the control.