Pkc412

MCF-7 cells were seeded in 96-well plates overnight and then transfected with pGL4.10-ckβ(−2000/−1) and pGL4.73[hRluc/SV40]. Six hours after transfection, the culture medium was replaced with 1% (v/v) serum-free DMEM for 20 hr before stimulation. The cells were treated with 10, 20, or 30 ng/mL PMA (EMD Chemicals, USA) for 6 hr, except for the time-course study, in which cells were treated with 20 ng/mL PMA for 6 to 48 hr. For controls, DMSO was added to the cells instead of PMA. For the identification of PKC isozyme involved in the PMA-mediated repression of the ckβ promoter, MCF-7 cells were individually or co-treated with PMA (20 ng/mL) and a PKC inhibitor [1 mM PKC412 (Tocris Bioscience, USA), 0.1 mM Go 6983 (Tocris Bioscience, USA), 10 µM PKCε inhibitor peptide (Santa Cruz, USA) or 10 µM PKCη pseudo-substrate inhibitor (Santa Cruz, USA)] for 6 hr. After the treatments, luciferase activities were measured using the Dual-Glo luciferase assay. To determine whether the PMA response element is localized to the Ets and GATA binding sites, pGL4.10-mut(Ets), pGL4.10-mut(GATA), and pGL4.10-mut(Ets/GATA) mutant constructs were individually transfected into MCF-7 cells, followed by incubation with 20 ng/mL PMA for 6 hr before luciferase activity was determined.