Foxm1 in Lung Tumors Figure 4. Foxm1 deletion from respiratory epithelial cells diminishes tumor cell proliferation. Lungs from epFoxm12/2 and control Foxm1fl/fl mice, that were given Dox in water starting 4 weeks before urethane injections, were photographed and fixed 28 weeks after the first urethane injection. Lung sections were used either for in situ hybridization or immunohistochemistry. A–B. In situ hybridization demonstrates that Foxm1 mRNA is selectively decreased in lung tumors of epFoxm12/2 lungs (B) versus control epFoxm1fl/fl mice (A). C–D. Diminished cell proliferation in epFoxm12/2 lung tumors (D) is shown using Ki-67 antibody. E. Decreased number of Ki-67-positive cells in epFoxm12/2 mouse lungs. Ki-67- positive cells were counted in ten random microscope fields from tumor regions of control and epFoxm12/2 lungs (mean+s.d.). Magnification: A panels, 106; B–D panels, 1006. doi:10.1371/journal.pone.0006609.g004 interfering RNA (siRNA) specific to the human Foxm1 mRNA (siFoxm1) or with mutant control siFoxm1 [13]. Forty-eight hours after siRNA transfection, total RNA was prepared from the A549 cells and analyzed for Foxm1 expression by qRT-PCR. siFoxm1 transfection efficiently reduced Foxm1 mRNA, also inhibiting the expression of Foxm1-target cyclin B1 gene (Fig. 9A). Consistent with our in vivo studies (Fig. 5A), Foxm1-depletion in vitro significantly decreased TOPO-2a mRNA, whereas PPARa mRNA was increased (Fig. 9A). Furthermore, protein levels of TOPO-2a were decreased after Foxm1 depletion in A549 cells as demonstrated by Western blot analysis (Fig. 9B). Lung epithelial-specific ablation of Foxm1 both in vivo and in vitro resulted in significant decrease in TOPO-2a mRNA expression, which suggests that Foxm1 plays direct or indirect role in the regulation of TOPO-2a gene expression. Since TOPO-2a is a prominent target for anti-tumor therapy [28] due to its critical role in tumor cell proliferation, we focused our next experiments to determine whether TOPO-2a gene is a direct transcriptional target of Foxm1. Three potential Foxm1 protein binding sites were identified in the –1.5 Kb promoter region of the mouse TOPO-2a gene (Fig. 9C). We next used Chromatin Immunoprecipitation (ChIP) assays to determine whether Foxm1 protein directly binds to the -1.5 Kb TOPO-2a promoter region in the context of endogenous DNA. The cross-linked and sonicated chromatin from untransfected mouse lung epithelial 15 (MLE-15) cells or MLE-15 cells transfected with Foxm1-specific siRNA was immunoprecip- PLoS ONE | www.plosone.org 6 August 2009 | Volume 4 | Issue 8 | e6609