Figure 7.

In SkBr3 and BT20 breast cancer cells, EGF engages E2 through GPR30 to boost the growth effects, which were monitored by MTT assay. A and E, The combination of E2 and EGF treatments enhances the proliferation of SkBr3 and BT20 cells stimulated by each mitogen used alone. Cells were treated with vehicle or 100 nm E2 and/or 50 ng/ml EGF in medium containing 2.5% charcoal-stripped FBS (medium was refreshed and treatments were renewed every 2 d). B and F, The growth effects induced by E2 alone or in combination with EGF were abolished by GPR30 silencing in both SkBr3 and BT20 cells. Cells were transfected with an empty vector or shGPR30 and the next day were treated with vehicle (−), 100 nm E2, and/or 50 ng/ml EGF. Transfections and treatments were renewed every 2 d. Efficacy of GPR30 silencing was evaluated by immunoblots, as indicated. C and G, The DN/c-fos construct effectively blocked the AP1 mediated transcriptional activity in SkBr3 and BT20 cells. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activity induced by 50 ng/ml EGF was calculated. D and H, The growth effects induced by E2 and EGF used alone or in combination were abolished transfecting the SkBr3 and BT20 cells with the DN/c-fos expression vector. Cells were transfected with an empty vector or the DN/c-fos construct and the next day were treated with vehicle (−), 100 nm E2, and/or 50 ng/ml EGF. Each data point is the mean ± sd of three independent experiments performed in triplicate.