Foxm1 in Lung Tumors Figure 9. Foxm1 transcription factor directly binds to and induces the mouse TOPO-2a promoter region. A. Foxm1 mRNA depletion in A549 lung adenocarcinoma cells led to decreased TOPO-2a and cyclin B1 mRNAs expression. A549 cells were mock transfected (control) or transfected with short interfering RNA (siRNA) duplex specific for Foxm1 mRNA (siFoxm1) or with control mutant siFoxm1 duplex. Forty-eight hours after siRNA transfection, total RNA was extracted and analyzed for Foxm1, TOPO-2a, PPARa, PPARc and cyclin B1 mRNAs by qRT-PCR. B. Western blot shows decreased protein levels of TOPO-2a and Foxm1 in siRNA-transfected A549 cells. C. A schematic drawing of the –1.5 Kb promoter region of the mouse TOPO-2a gene. Locations of three potential Foxm1 DNA binding sites are indicated (white boxes). D. ChIP assay demonstrated that Foxm1 protein binds to promoter regions of the TOPO-2a gene. Foxm1 binding to genomic DNA was normalized to IgG control antibodies. Diminished binding of Foxm1 to the endogenous mouse promoter regions of the TOPO-2a gene was observed after siFoxm1 transfection in MLE-15 cells. E. Foxm1 induced the transcriptional activity of TOPO-2a promoter. MLE-15 cells were transfected with CMV-Foxm1b expression vector and luciferase (LUC) reporter driven by the -1.5 kb mouse TOPO-2a promoter region. CMV-empty plasmid was used as a negative control. Cotransfection of CMV- Foxm1b and Foxm1-Luc reporter plasmids were used as a positive control. Cells were harvested at 24 hr after transfection and processed for dual LUC assays to determine LUC activity. Transcriptional activity of the mouse TOPO-2a promoter was increased by CMV-Foxm1b transfection. A p value,0.05 is shown with asterisk (*). doi:10.1371/journal.pone.0006609.g009 PLoS ONE | www.plosone.org 10 August 2009 | Volume 4 | Issue 8 | e6609