Figure 4.

 Cyclic AMP response element (CRE)‐site and CRE‐binding protein (CREB)‐mediated activation of human Arm protein lost in epithelial cancers, on chromosome X1 (ALEX1) promoter by the glycogen synthase kinase‐3 (GSK‐3) inhibitor. (a) Luciferase reporter assay of PANC‐1 cells transfected with wild‐type ALEX1 promoter‐driven reporter plasmid with/without the intron 3‐exon 4 region containing five TBE sites (+2156 to +3946). The bar graph represents the relative luciferase activities. (b) The left diagram is shown as described in the legend for Figure 1(b). The right bar graph represents the relative luciferase activities in control, 1‐mehyl‐6‐bromoindirubin‐3′‐oxime (MeBIO)‐, and 6‐bromoindirubin‐3′‐oxime (BIO)‐treated PANC‐1 cells transfected with the reporter plasmid driven by wild‐type, a series of 5′ deleted type, or a series of the site‐directed mutant type of human ALEX1 promoter. The error bars indicate the SD. (c) Luciferase reporter assay of PANC‐1 cells co‐transfected with wild‐type ALEX1 promoter‐driven reporter plasmid and expression plasmid of the indicated genes. The bar graph represents the mean ratios between the luciferase activities in PANC‐1 cells treated with BIO and MeBIO (BIO/MeBIO ratio), and the error bars represent the SD. *P < 0.05 compared to control (empty). (d) Chromatin immunoprecipitation followed by quantitative real‐time PCR was carried out with normal rabbit IgG or anti‐CREB antibody using the PANC‐1/empty clone 1 and PANC‐1/β‐catenin‐ΔS45 clone 1. Sequences of cyclin D1 promoter and 3′‐distal region of the ALEX1 gene were used as a positive and negative control for CREB binding, respectively.