The renilla luciferase reporter construct pRL-TATA was constructed by inserting a minimal TATA box of the adenovirus type 2 major late promoter [46] between the BglII and SalI sites of the pRL-null plasmid. Eukaryotic expression plasmids were generated by inserting PCR amplified full length cDNA of NF-YA (NM_002505.4), NF-YB (NM_006166), NF-YC (NM_014223.4), FOXO3 (BC058662) and FOXO4 (BC106761) into the EcoRV site of the pcDNA3 vector. Primers used for PCR amplification are listed in Table S1. FOXO3 A3 was created by mutating the three Akt/PKB phosphorylation sites T32, S253 and S315 to A and FOXO4 A3 has been generated by site directed mutagenesis of amino acid residues T32, S197 and S262 to A using primers listed in Table S1. All plasmids used in transient transfections were purified with the QIAGEN Plasmid Maxi Kit (Qiagen) and verified by sequencing prior to transfections. Luciferase Assays One day before transfection, C33a and ‘‘444’’ cells were plated at a density of 2.56104/well on a white Nunclon F-96-well plate (Nunc, Roskilde, Danmark). For the deletion- and substitution mutant analysis, cells were transfected with 250 ng/well of LKB1- promoter pGL3 firefly luciferase reporter and 25 ng/well of pRL- TK renilla luciferase plasmid for normalization using 0.9 ml/well of Lipofectamine 2000 (Invitrogen) for C33a cells and 1.2 ml/well for ‘‘444’’ cells. 48 h after transfection, cells were lysed, firefly luciferase activity was analysed and normalized to renilla luciferase activity using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. For co-expression of transcription factors and LKB1-luciferase reporters, cells were transfected with 100 ng/well of LKB1-promoter driven pGL3 firefly luciferase reporters, 0.5 ng/well of pRL-TATA normalisa- tion plasmid and 150 ng/well of the corresponding expression plasmid. All experiments were performed at least 3 times in quadruplicates. A one-way analysis of variance (ANOVA) using the GraphPad PRISMH program Version 5.0 followed by a Newman-Keuls post-hoc test was performed for statistical analysis of the results shown in Figure 1 and 2, and a two-way ANOVA followed by Bonferroni analysis was performed for statistical analysis of the results shown in Figure 7. Differences with a p value,0.05 were considered statistically significant. Electrophoretic mobility shift assay (EMSA) 200 ng of annealed synthetic oligonucleotide probes were end- labeled with 6000 Ci/mmol [c-32P]ATP (Perkin Elmer, Bosten, USA) by 10 U of T4 polynucleotide kinase (New England Biolabs) in a final reaction volume of 10 ml for 30 min at 37uC and purified from a 15% non-denaturing polyacrylamide gel. Approximately 0.2 ng (10–15000 cpm) of the probe was incubated together with either 25 ng of full length GST-tagged FOXO3 protein (Abnova, Taipei, Taiwan) or 2–4 mg of nuclear cell extracts for 30 min at 25uC in a final volume of 20 ml binding buffer containing 10% glycerol, 12 mM HEPES, pH 7.9, 4 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM dithiothreitol, 0.6 mg/ml bovine serum albumin, 0.5 mg of poly(dI-dC) (Sigma) and competitor as indicated. For supershift assays, 2 mg of the corresponding antibody were added after 30 min and incubation was continued for 1 h at 4uC. Subsequently the binding reaction was separated on a 5.5–7% polyacrylamide gel in 1X TB 90 mM Tris, 90 mM boric acid). Chromatin Immunoprecipitation (ChIP) ChIP assays were performed as described previously [47]. Briefly, 16107 of ‘‘444’’ cells were cross-linked with 1% Transcriptional Regulation of LKB1 formaldehyde for 10 min. The reaction was stopped by the addition of 0.125 mM glycine. Subsequently, cells were washed with phosphate-buffered saline (PBS) and lysed in ChIP-lysis buffer (5 mM HEPES pH 8, 85 mM KCl, 0.5% (v/v) Nonidet P-40, supplemented with 1 mM DTT, 5 mM NaF, 0.1 mM Na3VO4 and ‘‘Complete protease inhibitors’’ (Roche). Lysates were centrifuged to remove debris and resuspended in ChIP-buffer (20 mM Tris pH 8, 1.2 mM EDTA, 200 mM NaCl, 0.01% SDS, 1.1% Triton X-100) supplemented with 1 mM DTT, 5 mM NaF, 0.1 mM Na3VO4 and ‘‘Complete protease inhibitors’’(Roche). Then chromatin was sonicated to shear DNA to an average size of 500 bp, insoluble debris was removed by centrifugation and lysates (equivalent to 25 mg of DNA) were precleared adding 40 ml of protein A/protein G-agarose mixture (Roche) blocked with 40 mg of sheared salmon sperm DNA (Eppendorf) for 2 h at 4uC in a total volume of 500 ml ChIP-dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% (v/v) Triton X-100 and 0.01% (v/v) SDS). After centrifugation, supernatants were incubated with 10 mg of the corresponding antibody for 14 h and protein-DNA complexes were precipitated for 3 h with 40 ml of protein A/protein G-agarose mixture. After extensive washing, the immunocomplexes were eluted in 250 ml of 0.1 M NaHCO3 containing 1% (v/v) SDS and cross-links were reversed by adding NaCl to a final concentration of 200 mM and vigorous mixing at 65uC for 14 h. After protein digestion with proteinase K (New England Biolabs), DNA was recovered by phenol-chloroform extraction and ethanol precipitation and analyzed by semiquan- titative PCR using the forward primer (59-GTGACCTAC- GACCCCCTTC-39) and the reverse primer (59-GCTGAC- GATTGGAGCGTTTG-39) to amplify the LKB1 promoter region. Primers used for amplification of the LKB1 coding region encompassing Exon 4–5 were: forward (59-TCAGCTGATT- GACGGCCTGGA-39) and reverse (59-CCAGCCGACCA- GATGTCCAC-39). RNA Interference Small interfering RNA (siRNA) targeting NF-YA mRNA [48] (sense strand, 59-GUCCAGACCCUCCAGGUAG-dTdT-39; an- tisense strand, 59- CUACCUGGAGGGUCUGGAC-dTdT-39) or mRNAs of all FOXO family members [49] (sense strand, 59- AGGAUAAGGGCGACAGCAA-dTdT-39; antisense strand, 59- UUGCUGUCGCCCUUAUCCU-dTdT-39) and non-targeting control [50] (DNA target sequence, 59-AACAGTCGCGTTTGC- GACTGG-39) were purchased from Qiagen. Cells were transfect- ed using the HiPerfect reagent (Qiagen) according to the manufacturer’s instruction. For transfection-complex formation, 0.4 nmol of siRNA were mixed with 20 ml HiPerfect in 100 ml of serum free DMEM. After 10 min of incubation, C33a and ‘‘444’’ cells were seeded at a density of 56105/6 cm plate, IMR-90 at a density of 7.56105/6 cm plate in 4 ml of normal growth medium and 100 ml of the transfection-complex were added dropwise to the cell-suspension, leading to a final concentration of 100 nM siRNA in the culture medium. 48 h after transfection, cells were splitted and an identical second transfection was performed. 96 h after the first transfection, cells were lysed as described previously [30] and cytoplasmic RNA was isolated using the RNeasy Kit (Qiagen). cDNA was obtained from reverse transcription of 1 mg of total RNA using 10 ng/ml of p(dN)6 random primers (Roche) and SuperScript II reverse transcriptase (Invitrogen) in a final volume of 20 ml according to the manufacturer’s instructions. 1 ml of cDNA was subsequently used for PCRs using Platinum Taq DNA polymerase (Invitrogen). All PCRs were monitored within the linear range, which has been determined for each reaction individually. PCR condidions for each reaction, including primers, PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e32590