FIG. 4.

Two pulses of endosomal EGFR signaling are sufficient to stimulate cell proliferation. Proliferation was induced from two temporally separate endosomal EGF pulses. MDCK and BT20 cells were plated at 10,000 cells per coverslip, starved for 36 h, and then treated with AG1478 (AG) and EGF (100 ng/ml), with or without monensin (Mon), followed by washing and incubation in starvation medium. Some cells were left until the end of the assay, while others were treated as described above a second time for 4, 8, or 12 h. Cells treated without monensin were instead stripped of plasma membrane-recycled ligand prior to EGFR activation. At 18 h, cells were fixed and assayed for BrdU incorporation. Cells were counted at 300 per sample, and data were plotted as the mean of triplicate experiments.