<p>(A) Wild type BRCA1 activates the p3TP-Lux promoter. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase plasmids were co-transfected with Flag-Smad3 and HA-BRCA1 plasmids into COS-7 cells, as indicated. A pcDNA3 empty vector plasmid was used to adjust total DNA amounts. At 36 hrs after transfection, luciferase and <i>β</i>-galactosidase assays were performed. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. *, <i>P</i><0.05 versus empty vector only. (B) Wild type BRCA1 enhances TGF-<i>β</i>-mediated p3TP-Lux promoter activity. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase plasmids were co-transfected with 0.25 µg of caT<i>β</i>RII plasmid or 0.1 µg of HA-BRCA1 plasmid into COS-7 cells, as indicated. 24 hrs after transfection, the indicated cells were incubated with 2 ng/ml of TGF-<i>β</i>1 ligand overnight, followed by luciferase and <i>β</i>-galactosidase assays. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. (C) BRCA1 mutants could not activate the p3TP-Lux promoter. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase were co-transfected with 0.1 µg of wild type or various mutant forms of HA-BRCA1 into COS-7 cells. 24 hrs after transfection, the cells were treated with or without 2 ng/ml of TGF-β1 overnight, followed by luciferase and <i>β</i>-galactosidase assays. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. WT: wild type BRCA1; M, P, and Y: BRCA1-M1775R, BRCA1-P1749R, and BRCA1-Y1853x, respectively; N-: BRCA1 (1–683aa); C-: BRCA1 (1301–1863aa).</p
