FIGURE 3.

Smad7 regulates transcription of caspase 8. A, to understand the mechanism of induced expression of caspase 8 in MCF7-Smad7 cells, we performed the luciferase assays using caspase 8 promoter-luciferase containing the promoter region (−1615/+16). Each cell was transfected with luciferase DNA using Lipofectamine, and relative luciferase activity was measured using Victor² luminometer. The relative activity was normalized using β-galactosidase activity. B, caspase 8 luciferase construct was cotransfected with increasing amounts of Smad7 expression construct in MCF7 cells. The luciferase activity was measured with a luminometer after 36 h, and the expression level of Smad7 was detected with Western blotting. C, to identify the regulatory region of caspase 8 promoter, the deletion constructs were generated as shown in D. With deletion constructs of the caspase 8 promoter, the caspase 8 reporter assay was performed to minimize the potential regulatory region. After transfection of reporter constructs with increasing amounts of Smad7, luciferase activity was measured after 36 h and normalized with β-galactosidase activity. (n = 3 independent experiments; error bars represent S.D.)