Figure 5.

DIM-C-pPhOCH₃-dependent activation of the p21 promoter and recruitment of Nur77 to the promoter. DIM-C-pPhOCH₃ induced transactivation in cells transfected with pWWP (A) and its mutants (B). Panc1 cells were transfected with pWWP and its mutants, and treated with DMSO or DIM-C-pPhOCH₃ (A and B). Cells were also transfected with pWWP101 and siNur77, siSp1, or siScr, and treated with DMSO or DIM-C-pPhOCH₃ (B). Luciferase activity (relative to β-galactosidase activity) was determined as described in the Materials and Methods. Significant (p < 0.05) induction by DIM-C-pPhOCH₃ (*) is indicated. (C) Sp protein knockdown. Panc1 cells were transfected with siScr, siSp1, siSp3 or siSp4, treated with DMSO or DIM-C-pPhOCH₃, and after 24 hr, whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. Results (A and B) are expressed as means ± SE of triplicate experiments and significant (p < 0.05) induction by DIM-C-pPhOCH₃ (*) or inhibition of induction by RNA interference (**) are indicated. (D) Time course chromatin immunoprecipitation assay. Panc1 cells were treated with 10 μmol/L DIM-C-pPhOCH₃ for 1, 2 and 6 hr, and interactions of Sp1, Sp3, Sp4 and Nur77 with the p21 promoter were determined as described in the Materials and Methods.