FIG. 3.

Two pulses of standard EGFR signaling are sufficient to stimulate cell proliferation. (A) Proliferation induced from a continuous mitogenic pulse. MDCK and BT20 cells were plated at 10,000 per coverslip and serum starved for 36 h. Cells were then stimulated for various times with either FBS (10%) or EGF (100 ng/ml) in the presence of 25 μM BrdU, after which unbound ligand was removed and cells were incubated in starvation medium. At 18 h, cells were fixed and assayed for BrdU incorporation. (B) Proliferation induced from two temporally separate EGF pulses. Cells were plated and starved as described above and then stimulated with EGF (or FBS) for the times indicated before removal of free ligand and culture once again in starvation media. A second pulse of growth factor was administered again at 4, 8, or 12 h and washed out after the times indicated. Cells were then fixed and assayed as indicated in panel A. Sample fields of MDCK and BT20 nuclei were quantitated by immunofluorescence of BrdU incorporation. A total of 300 cells per sample were counted, and data were obtained from triplicate experiments.