FoxM1 regulation of β-catenin transcription. (A) Schematic drawing of the 6-kb promoter region of the human ctnnb1 gene. FoxM1 binding sites are indicated (open box); the first exon is also show (shaded box). The three luciferase reporter constructs with various deletions of FoxM1 binding sites are also shown. (B) ChIP assay demonstrating that FoxM1 directly binds to two promoter regions of the ctnnb1 gene. Cross-linked chromatin from either mock-transfected or FoxM1 siRNA- or scRNA- transfected HMVEC-L was immunoprecipitated with either anti-FoxM1 antibody or IgG control. After immunoprecipitation, genomic DNA was analyzed for the amount of ctnnb1 promoter DNA using quantitative real-time PCR with primers specific for each region. FoxM1 binding to genomic DNA was normalized to IgG control. Data are shown as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus either mock or scRNA. (C) Luciferase reporter assay demonstrating that FoxM1 induced the transcriptional activity of the human ctnnb1 gene. After transfection with either luciferase reporter constructs under the control of a promoter of the human ctnnb1 gene with indicated deletions (a, b, and c) or empty vector (vector), HMVEC-L were plated at subconfluent (40%) or confluent conditions. At 40 h after transfection, the cells were collected for analysis of luciferase activity. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus either construct a or b at a confluent condition; #, P < 0.01 confluent versus subconfluent conditions; **, P < 0.005 versus either construct a or b at a subconfluent condition. (D) Western blot analysis demonstrating markedly decreased FoxM1 expression in confluent HMVEC-L. Cell lysates of HMVEC-L at 100% confluency and 50–70% confluency were used for Western blotting of FoxM1 expression. The experiment was performed three times with similar results.