Fig. 3.

ErbB1 kinase inhibitor, AG1478, and anti-estrogen, ICI 182780 (ICI), block the combined effects of estrogen and EGF on cell proliferation and stimulation of PRL gene expression. GH3 cells transiently cotransfected with PRL reporter gene and control reporter gene were treated with vehicle (C), E2 (0.01 nM), EGF (5 ng/ml), or a combination of E2 and EGF (EE), either alone or in presence of ICI 182780 (100 nM) (A) or AG1478 (10 μM) (B) for 24 h, and normalized luciferase activity was determined as described in materials and methods. Data were calculated as fold change over control (arbitrary value of 1). Each value is the mean ± SE of 3 separate experiments, each performed in triplicate. *Significant difference from control; **significant difference from E2 and EGF alone; ***significant difference from treatments without ICI/AG1478; ^ano significant difference from E2 alone, (P < 0.05). C: GH3 cells were treated with vehicle (C), E2 (0.01 nM), EGF (5 ng/ml), or EE, either alone or in the presence of AG1478 (10 μM) or ICI 182780 (100 nM), and cell proliferation was determined by the MTT assay after 5 days. Data were calculated as percentages of vehicle control and presented as the means ± SE of 3 separate experiments. *Significant difference from control, **significant difference from E2 and EGF alone, ***significant difference from treatments without ICI, ****significant difference from treatments without AG1478; ^ano significant difference from E2 alone (P < 0.05). D: GH3 cells were treated with vehicle or ICI 100 nM for 18–24 h, and an equal amount of cell lysates was subjected to Western blotting with an anti-ERα antibody, followed by stripping and reprobing with anti-ERβ antibody, followed by stripping and reprobing with anti-actin antibody. Results shown are from a single experiment and are representative of 3 separate experiments.