Compound supply and recombinant proteins
All chemicals were purchased from commercial sources except for the PI3K inhibitor PI-103, which was synthesized following published patent specifications. Cisplatin was provided by C. Navarro, Minerval was generously provided by P. Escriba, and all other chemicals were purchased from commercial sources: LY294002, Ratjadone A, were purchased from Calbiochem (San Diego, CA); Forskolin, Leptomycin B and Rapamycin, were purchased from LC Laboratories (Woburn, MA, U.S.A.); DMSO was purchased from Sigma-Aldrich (St. Louis, USA); Epidermal growth factor (EGF), platelet-derived growth factor (PDGF) were purchased from RELIATech A.S. (Braunschweig, Germany); and human Insulin-Like Growth Factor-I (IGF-I) and human insulin were purchased from (Roche Diagnostics, Mannheim, Germany). Stock solutions of the test compounds were deposited in three different concentrations in 96-well master plates, transferred to multiple replica plates and frozen at -80°C.
The luciferase reporter constructs pGL-1xDBE, pGL-2xDBE, pGL-3xDBE, pGL-4xDBE, pGL-5xDBE and pGL-6xDBE were generated by inserting one to six copies of the DBE consensus sequence  in front of a SV40 minimal viral promoter of the pGL3-Promoter vector (Promega). The annealed and phosphorylated oligonucleotides 5'-CTAGAAGTAAACAA-3' (1xDBE-forward) and 5'-GATCTT-GTTTAC-3' (1xDBE-reverse) or 5'-CTAGAAGTAAACAACTATGTAAACAA-3' (2xDBE-forward) and 5'-GATCTTGTTTACATAGTTGTTTACTT-3' (2xDBE-reverse) were ligated as single copy or concatemerized into NheI and BglII digested pGL3 promoter vector. In order to generate the negative control plasmid pGL-3xDBEmut, three copies of a DBE sequence that contains a point mutation that prevents FOXO binding (annealed and phosphorylated oligonucleotides (3xmDBE-forward) 5'-CTAGAAGTA-AGCAACTATGTAAG-CAACTATGTAAGCAA-3' and (3xmDBE-reverse) 5'-GAT-CTTGCTTACATAGTTGCTTACATAGTTGCT-TACATAG-3') were inserted into pGL3-Promoter vector. The constitutively active construct FOXO3a-A3, in which three PI3K-dependent phosphorylation sites have been mutated to alanine was kindly provided by Dr. M. Hu (University of Texas M. D. Anderson Cancer Center, Houston). In order to generate stable cell lines for the assay, we inserted a puromycin-resistance cassette into the pGL-3xDBE construct. This was achieved by PCR amplifying the puromycin resistance gene from pBABEpuro vector and cloning it into the SalI and BamHI sites of the pGL3-Promoter vector derived pGL-3xDBE construct, thereby obtaining pGLpuro-3xDBE.
U2-OS cells obtained from the ATCC were cultivated in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS, Sigma), antibiotics and antimycotics in a humidified incubator at 37°C with 5% CO2. U2foxRELOC cells have been described previously .
Transfection and Luciferase assays
U2OS cells were cultured in a 96-well plate (100 μl final volume per well) and transfected at 70% confluence with the plasmids indicated using the effectene transfection reagent (Qiagen). LY294002 or insulin were added individually to wells 42 hours later, at a final concentration of 20 μM or 5 mg/ml, respectively, and the cells were incubated for an additional 6 h. Luciferase assays were carried out using the Dual-Luciferase Reporter Assay System (Promega), according the manufacturer's instructions on a multilabel plate reader (Wallac Victor, Perkin-Elmer), and the ratio of firefly- to Renilla-luciferase activities was calculated. All values were presented as means ± SEM. The unpaired t-test (two-tailed) was performed for statistical analysis using the GraphPad PRISM® Version 4.0 program. Differences with a p value < 0.05 were considered statistically significant.
Generation and maintenance of U2transLUC cells
U2foxRELOC cells were transfected at 75% confluence with pGLpuro-3xDBE using the effectene transfection reagent (Qiagen). Cells were selected with puromycin (Calbiochem) for five days and the resistant colonies that best expressed the reporter constructs were then recovered and cultured, as was the most homogeneous population. Fluorescence-activated cell sorting (FACS) of GFP-FOXO expressing cells was performed on a FACSAria (BD Biosciences, San Jose, CA, USA). U2transLUC cell clones were maintained in DMEM, supplemented with 10% FBS (Sigma), antibiotics and antimycotics, 0.1 mg/ml Neomycin and 1 μg/ml puromycin. Cell cultures were maintained in a humidified incubator at 37°C with 5% CO2, and they were passaged when confluent using trypsin/EDTA.
Crystal violet assay
Triplicate samples of 104 cells were seeded in 2.5 cm dishes and allowed to attach. After 24 hours, the medium was removed and replaced with culture medium with sodium azide at the concentrations indicated, while the controls remained untreated. After the appropriate time period the cells were fixed with 0.5% glutaraldehyde and stained with 1% crystal violet. After extensive washing, crystal violet was resolubilized in 10% acetic acid and quantified at 595 nm as a relative measure of cell number.
Viability assay measuring fluorescent intensity
Samples of 104 cells per well were allowed to attach overnight in 96-well Greiner plates and the following day the culture medium was replaced with fresh medium containing the concentrations of sodium azide indicated. After a three hour treatment, the cells were washed with PBS and the fluorescent intensity was measured in a multilabel plate reader (Wallac Victor 2, Perkin-Elmer) using UV light as the excitation source, as well as a F485 CW lamp Filter and a F535 CW emission filter for 0.5 seconds per well. The values are the averages obtained from experiments carried out in triplicate.
U2transLUC cells were seeded at a density of 1.0 × 105 cells/ml in black-wall clear-bottom 96-well microplates (BD Biosciences) using a Titan Multidrop 384 automatic dispenser (Titertek Instruments, Inc., Huntsville, AL). The final volume of the cell suspension was 200 μl in each well. After incubation at 37°C in 5% CO2 for 12 hours, 2 μl of each test compound was transferred from the master plate to the assay plate. Cells were incubated in the presence of the compounds for 1 hour and the far-red fluorescent cell-permeable DNA probe, DRAQ5™ (Biostatus Ltd, Leicestershire, UK), was then added to all wells at a final concentration of 5 mM 15 minutes prior to obtaining the images. The images were acquired as described previously  using a BD Pathway™ 855 Bioimager equipped with a incubation chamber that provided a constant temperature of 37°C in 5% CO2. Images were acquired in the GFP and DRAQ5 channels using 488/10 nm EGFP excitation filter, a 515LP nm EGFP emission filter and 635/20 nm/695/55 nm DRAQ5 excitation/emission filter with a 10× dry objective. The plates were exposed for 0.066 ms (Gain 31) to acquire DAPI images and 0.55 ms (Gain 30) for GFP images. Image and data analysis was performed as described previously . After image acquisition, the plates were incubated for another 5 hours at 37°C and then the average GFP intensity per well was measured as described above. Finally, U2transLUC cells were processed to measure the firefly luciferase activity using the Luciferase Assay System (Promega) on a multilabel plate reader (Wallac Victor, Perkin-Elmer) according to the manufacturer's instructions. The relative luciferase activity, as a measurement of FOXO3a's transcriptional activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the the average GFP intensity from the same well.