Figure 4.
Cd activates target gene expression through ERα. MCF-7 cells were transfected with either Ri-ERα (black) or Ri-Control (white) along with (panel A) CycD1-luc or (panel B) c-myc-luc. Renilla luciferase construct was cotransfected as an internal control. After transfection, cells were treated with 10−6m CdCl2, PPT, 10−6m plus PPT, or mock treated [control (C)]. Luciferase activity was measured using a dual luciferase assay. Statistics represent triplicate samples. Panel C, MCF-7 cells were hormone deprived and treated with 10−6m CdCl2 (Cd) or mock treated with PBS (C), followed by fixation in formaldehyde. ChIP analysis was performed with α-ERα antibodies, and ERα recruitment to target gene promoters was determined by promoter-specific primers and semiquantitative RT-PCR. Primer positions are depicted on the promoter diagram for each gene. Input represents total chromatin before immunoprecipitation. Panel D, Band intensities of PCR product were quantitated using Quantity One software (Bio-Rad) and statistics represent fold changes of three independent experiments.