Determination of cell numbers after EGF stimulation. (A) A549 and HK2 cells were cultured in medium containing 10% FCS (control), 2.5% FCS or 2.5% FCS plus EGF (100 ng), and the cells were counted after 24, 48 and 72 hours. Five thousand cells from each group were analyzed in all experiments. (B) The cells were cultured in medium containing 10% FCS with or without EGF (100 or 200 ng) supplementation. The cells were counted after 48 hours. In all experiments, 5000 cells from each group were analyzed. (C) The cells were fixed after 48 hours of treatment (C10%FCS, 10%FCS + 100ngEGF, C2.5%FCS, 2.5%FCS + 100ngEGF), and the metaphases were quantified in a total of 2000 cells. All results are representative of three or more independent experiments. *p ≤ 0.05. Bars = standard deviation.