Fig. 3. Effects of G3EGF on cell growth under different culture conditions. (A) Cells (2x10⁵ cells per well) were seeded on six-well culture plates maintained in DMEM containing 10% FBS. 1 hour after cell inoculation, G3EGF-transfected U87 cells exhibited spreading on the plates, but vector-transfected U87 cells had only started to attach. Scale bars, 40 µm. (B) Serum was withdrawn from the cultures by changing the culture medium to DMEM alone. At 24 hours, most of the vector-transfected cells had rounded and some had developed long processes to connect to each other, but the G3EGF-transfected cells remained spread on the plates. (C) Cells before and after serum withdrawal were harvested at different time intervals and cell numbers were counted to obtain a growth curve. The total number of the vector-transfected cells declined slowly. However, the G3EGF-transfected cells continued to grow for 5 days, followed by dramatic cell detachment and cell death. (D) Cell death was determined by trypan-blue staining. G3EGF transfection enhanced cell death after serum withdrawal. (E) Cultured in serum-free DMEM, the vector-transfected cells could be maintained for 4 weeks, whereas the numbers of G3EGF-transfected cells declined dramatically owing to cell death. All the experiments were repeated four times. *P<0.01.