Figure 4

Pokemon stimulates survivin expression by direct binding to its promoter. (A) Schematic representation of survivin promoter-luciferase reporter plasmids, including wild-type survivin promoter and 5' progressive deletions, as well as site-directed mutants at Pokemon binding sites, pLuc-95 m (at -95 bp) and pLuc-231 m (at -231 bp). Refer to Materials and methods for the plasmid construction. Survivin promoter activity was analyzed in MCF-7 cells by luciferase assay. (B) Regulation of survivin promoter activity by Pokemon in MCF-7 and MDA-MB-231 breast cancer cells. Wild-type and mutant promoter (pLuc-441) was cotransfected with Pokemon gene or Pokemon small interfering RNA siRNA#1, and luciferase activity was measured with β-galactosidase as an internal control. Data represent the mean of three independent experiments ± SD. Statistical significance was tested using Student's t-tests with *P < 0.05 and **P < 0.01. (C) Chromatin immunoprecipitation assay indicating that Pokemon bound to survivin promoter in MCF-7 and MDA-MB-231 breast cancer cells. Rabbit immunoglobulin G was used as a negative control. Refer to Materials and methods for details.