mouse TCF-4, Mm00501505_m1; mouse Cyclin D1, Mm00432359_m1; mouse Vegfa, Mm00437304_m1; mouse TTF- 1, Mm00447558_m1; human FoxM1, Hs00133543_m1; human Cyclin B1, Hs00259126_m1; human PPARa, Hs00231982_m1; human PPARc, Hs00234592_m1; human TOPO-2a, Hs01032127_g1; human b–Actin, Hs99999903_m1. Reactions were analyzed in triplicates and expression levels were normalized to b- Actin. siRNA transfection We transfected 100 nmol/L of either Foxm1-specific siRNA (siFoxm1; Dharmacon Research, Lafayette, CO) or mutant control siFoxm1 into human A549 lung adenocarcinoma cells (purchased from ATCC) using LipofectamineTM 2000 reagent (Invitrogen) in serum free tissue culture media as described previously [13,20]. Forty-eight hours after transfection, the A549 cells were used to prepare total RNA. Western blot Nuclear protein extracts were prepared from A549 adenocar- cinoma cell lines at 48 hours following siRNA transfection or mock transfection using Nuclear/Cytosol Fractionation kit (K266- 100, BioVision, Mountain View, CA) following protocols provided by the manufacturer, and Western blot analysis was done as described previously [13]. The rabbit anti-Foxm1 antibody (1:1000; clone C-20; Santa Cruze Biotechnology, Santa Cruze, CA) and mouse anti-TOPO-2a antibody (1:2500; cat# MAB4197; Chemicon) were used. The signals from the primary antibody were amplified by HRP-conjugated anti-mouse IgG (Bio- Rad, Hercules, CA) and detected with Enhanced Chemilumines- cence Plus (Amersham Pharmacia Biotech, Piscataway, NJ) followed by autoradiography. Cotransfection studies and chromatin immunoprecipitation (ChIP) assay We used PCR of mouse genomic DNA to amplify the -1544/ +17 region of the mouse TOPO-2a promoter (Gene Bank Number NT_165773.2) using following primers: 59-GGG GTA CCC CCC CCC AAA AAA AAA AAC ACC-39 and 59-CGA CGC GTA References 1. Toloza EM, Morse MA, Lyerly HK (2006) Gene therapy for lung cancer. J Cell Biochem 99: 1–22. 2. McCormick F (1999) Signalling networks that cause cancer. Trends Cell Biol 9: M53–M56. 3. Giaccone G (1996) Oncogenes and antioncogenes in lung tumorigenesis. Chest 109: 130S–134S. 4. Mitsuuchi Y, Testa JR (2002) Cytogenetics and molecular genetics of lung cancer. Am J Med Genet 115: 183–188. 5. Sherr CJ, McCormick F (2002) The RB and p53 pathways in cancer. Cancer Cell 2: 103–112. 6. Malkinson AM (1998) Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp Lung Res 24: 541–555. 7. Ye H, Kelly TF, Samadani U, Lim L, Rubio S, et al. (1997) Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol 17: 1626–1641. 8. Major ML, Lepe R, Costa RH (2004) Forkhead Box M1B (FoxM1B) Transcriptional Activity Requires Binding of Cdk/Cyclin Complexes for Phosphorylation-Dependent Recruitment of p300/CBP Co-activators. Mol Cell Biol 24: 2649–2661. 9. Ma RY, Tong TH, Cheung AM, Tsang AC, Leung WY, et al. (2005) Raf/ MEK/MAPK signaling stimulates the nuclear translocation and transactivating activity of FOXM1c. J Cell Sci 118: 795–806. 10. Costa RH, Kalinichenko VV, Major ML, Raychaudhuri P (2005) New and unexpected: forkhead meets ARF. Curr Opin Genet Dev 15: 42–48. 11. Krupczak-Hollis K, Wang X, Kalinichenko VV, Gusarova GA, Wang I-C, et al. (2004) The Mouse Forkhead Box m1 Transcription Factor is Essential for Hepatoblast Mitosis and Development of Intrahepatic Bile Ducts and Vessels during Liver Morphogenesis. Dev Biol 276: 74–88. Foxm1 in Lung Tumors AAG CGA CAA AAC CAG CGG-39. The PCR product was cloned into pGL3-Basic firefly luciferase (LUC) reporter plasmid (Promega) and then verified by DNA sequencing (Gene Bank Number NT_165773.2). We transfected mouse lung epithelial MLE-15 cells [37] with either CMV-Foxm1b (Gene Bank number NM_008021) or control CMV-empty expression plasmids, as well as with LUC reporters driven by –1.5 kb mouse TOPO-2a promoter. CMV-Renilla was used as an internal control to normalize transfection efficiency. Dual luciferase assay (Promega) was performed 24 hours after transfection as described [37]. Untransfected or siRNA-transfected mouse lung epithelial MLE-15 cells [37] were cross-linked by addition of formaldehyde, sonicated and used for the immunoprecipitation with Foxm1 rabbit polyclonal antibodies (H-300, Santa Cruz, CA) as described previously [25]. DNA fragments were 500 bp – 1000 bp. 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(2006) Identification of a chemical inhibitor of the oncogenic transcription factor forkhead box M1. Cancer Res 66: 9731–9735. 18. Kalinichenko VV, Major M, Wang X, Petrovic V, Kuechle J, et al. (2004) Forkhead Box m1b Transcription Factor is Essential for Development of Hepatocellular Carcinomas and is Negatively Regulated by the p19ARF Tumor Suppressor. Genes & Development 18: 830–850. 19. Gusarova GA, Wang IC, Major ML, Kalinichenko VV, Ackerson T, et al. (2007) A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment. J Clin Invest 117: 99–111. 20. Kim IM, Ackerson T, Ramakrishna S, Tretiakova M, Wang IC, et al. (2006) The Forkhead Box m1 Transcription Factor Stimulates the Proliferation of Tumor Cells during Development of Lung Cancer. Cancer Res 66: 2153–2161. PLoS ONE | www.plosone.org 12 August 2009 | Volume 4 | Issue 8 | e6609