Fig. 5. Effects of EGF on cell proliferation, apoptosis and EGFR phosphorylation. After serum withdrawal, cultures were maintained in the presence or absence of EGF (40 ng ml^–1). Cell numbers were counted at days –1 (cell inoculation in DMEM containing 10% FBS), 0 (serum withdrawal and EGF addition), 1, 3, 5 and 7. The addition of EGF had a greater effect on the growth of G3EGF-transfected U87 cells (A) than vector-transfected U87 cells (B). Data are expressed as the means±SD of three experiments each performed in triplicate. *P<0.01. (C) Cell apoptosis was analysed in the G3EGF-transfected cells. After serum withdrawal, the addition of EGF reduced cell apoptosis to 52% on day 7 compared with cells maintained in the absence of EGF (94% undergoing apoptosis). Cells cultured in DMEM/10% FBS were used as controls for the analyses (n=3). (D) After starvation, the cultures were treated with EGF (100 ng ml^–1) for 0, 5 or 20 minutes, as indicated. Cell lysate was prepared for western blotting probed with anti-phosphorylated-EGFR antibody. (Top) 1 minute exposure; (bottom) 30 minutes exposure. (E) The same amount of cell lysate was also analysed and probed with anti-EGFR antibody. G3EGF expression enhanced EGFR turnover (arrow). The same membrane was also probed with anti-actin antibody (arrow) to ensure equal loading of protein samples. Results presented in (D) and (E) were repeated twice.