Figure 4. Src is involved in EGF induction of cyclin D1.

(A). Western blot analysis of cyclin D1 expression in MDA-MB-231 and -436 cells. Cells were treated with vehicle (PBS) and EGF alone or together with the Src inhibitors PP2 and Dasatinib, the EGFR inhibitor AG1478 and PI3K inhibitor LY294002. Cell lysates were analyzed with anti-cyclin D1 antibody and anti-actin antibody was used to ensure equal loading. The experiment was repeated three times, and the representative results are shown. (B). Src inhibitors inhibit EGF induction of cyclin D1 promoter activity. ER-negative breast cancer cells were transfected with the luciferase reported plasmid cyclin D1 pl-963 that containing a luciferase gene driven by the cyclin D1 promoter. Transfected cells were treated with vehicle (PBS), 10 ng/ml or 500 ng/ml of EGF and 10 ng/ml EGF plus different inhibitors. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity in cells treated with vehicle that is arbitrarily set as 1.0 from four independent experiments; bars, SE. *, P<0.05, for cells treated with vehicle (PBS) vs 10 ng/ml of EGF. (C). The involvement of Src in EGF induction of cyclin D1 promoter activity. Cells were transfected with the luciferase reported plasmid cyclin D1 pl-963 together with an empty expression vector or Src mutants, a dominant-negative mutant (SrcK295R) and a constitutively active mutant (SrcY527F). Transfected cells were treated with vehicle (PBS), 10 ng/ml or 500 ng/ml of EGF. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity from four independent experiments. Luciferase activity in transfected cells treated with vehicle is arbitrarily set as 1.0; bars, SE. *, P<0.05, for cells treated with vehicle (PBS) vs 10 ng/ml of EGF.