Fig. 7

Mammea E/BB treatment attenuated WT1 - DNA binding to the proximal WT1 promoter and WT1 promoter activity. a K562 cells were treated with 3.5 μM mammea E/BB for 72 h and ChIPs were performed. Chromatin lysates were immunoprecipitated with antibodies to WT1, c-Fos/AP-1, Pol II (positive control) or IgG (negative control). ChIP lysates and 1:50 dilution of input were assayed by standard PCR using primers containing the consensus sequence for WT1 located at the WT1 proximal promoter. The WT1 immunoprecipitated lysates from mammea E/BB or vehicle control treatment were analyzed by SYBR green RT-PCR and graphed as relative DNA enrichment over 1:50 input as percentage of vehicle-treatment as shown in b. For WT1 promoter reporter activity, K562 cells were transfected with the pGL3_basic luciferase reporter (Luc) vector containing 301 bp of the wild type and mutant WT1 proximal promoter (c) followed by 1.5, 2.5, and 3.5 μM or vehicle treatment for 72 h. WT1or a mutant construct was used to transfect for 24 h and then assayed for firefly luciferase and β-galactosidase (β-Gal) activities. Site directed mutagenesis of the WT1 consensus sequence (-50 to -39) abrogated the WT1 promoter activity compared to the wild type WT1 promoter construct (301 bp WT1). The firefly luciferase and β-galactosidase activities were assayed and relative activities were graphed compared to the pGL3 basic vector (d). Experiments were performed a minimum of three times and representative graphs are shown. Data are the mean value ± SD of three independent experiments. Asterisks (*) denote values that were significantly different from the vehicle control (p < 0.05)