Cell culture and reagents
SW 480 (colon adenocarcinoma) and MCF-7 (breast cancer) cell lines were grown in DMEM (GibcoBRL, Life technologies, Cergy-Pontoise, France), supplemented with 10% FCS (Lonza, Levallois-Perret, France) 100 U/mL penicillin, 10 μg/mL streptomycin (GibcoBRL), 1 mM sodium pyruvate (GibcoBRL), MEM vitamins 100 × (GibcoBRL) and 5 μg/mL plasmocin (Cayla InvivoGen, Toulouse, France). The KG-1 cells were grown in 10% FCS supplemented IMDM medium (GibcoBRL). For the STAT3 overexpression experiments the plasmid PLZst3α was used. The STAT3 DNA binding domain (DBD)-mutant containing two mutations in the DBD that completely prevented DNA binding but allowed dimerization and nuclear entry, was a kind gift from Dr. C. Horvath (Northwestern University, Chicago, USA) . For some experiments, cells were treated with TNFα (20 ng/ml) (Sigma-Aldrich, Montigny le Bretonneux, France). To enhance STAT3 activation, cells were treated for 1 hr with IL-6 (50 ng/ml) (Sigma). Sodium orthovanadate (100 μM) (stock solution: 100 mM) was from Fischer (Illkirch, France), leptomycin B (LMB) (10 ng/ml) was from Sigma-Aldrich.
For cell infection with lentiviral shRNA, a set of two STAT1-targeting shRNAs that has previously been found to reduce the expression of STAT1  was used and transduced as previously described . Efficiency of infection was verified by measuring GFP by flow cytometry, and the efficacy of the inhibition of the shRNA's inhibition of STAT1 expression was verified by western blotting using a STAT1-specific antibody (Cell Signaling, Ozyme, St Quentin Fallavier, France).
For siRNA STAT3 silencing, the following double stranded siRNA oligonucleotide, previously shown to suppress STAT3 expression in a colorectal cell line , was purchased from Sigma-Aldrich: 5'-AACAUCUGCCUAGAUCGGCUAdTdT-3'; 3'-dTdTGUAGACGGAUCUAGCCGAU-5', along with a universal control set of siRNA (Sigma Aldrich). Cells (105 cells/well; density: 60%) were transfected using polyethylene imine (PEI) with 10 nM siRNA in culture medium without antibiotics. After 48 h or 72 h, cells were harvested and analyzed for annexin V binding by flow cytometry. In control cells, the irrelevant control siRNA was used. All experiments were performed in triplicate.
Preparation of subcellular fractions
Cells (20 × 106) were resuspended in cell lysis buffer containing 20 mM Hepes pH 7.4, 1 mM MgCl2, 10 mM KCl, 0.3% NP40, 0.5 mM DTT, 0.1 mM EDTA and protease inhibitors (CompeteTM, Boerhinger, France), and placed at 4°C for 5 min. The lysates were centrifuged at 14000 g for 5 min at 4°C, and the supernatant containing the cytoplasmic fraction was stored in aliquots at -80°C. The pellets were resuspended in cell lysis buffer adjusted to 20% glycerol and 0.35 M NaCl and placed at 4°C for 30 min. After centrifugation at 14000 g for 5 min at 4°C, the supernatant, containing the nuclear proteins, was stored at -80°C. Protein amounts were determined before use with the micro-BCA protein determination kit (Pierce, Perbio, Brebières, France).
The STAT3-decoy ODNs used were: RHN(CH2)6- CATTTCCCGTAAATCGAAGATTTACGGGAAATG -(CH2)3NHR (hp STAT3-decoy ODN), derived from the serum-inducible element of the human c-fos promoter , and RHN(CH2)6- CATTTGCCACAATCGAAGATTGTGGCAAATG -(CH2)3NHR (hairpin STAT3-decoy mutated ODN) (Sigma-Proligo) where R was either H, FITC or biotin. The decoy NF-κB-ODN consisted of: RNH(CH2)6-CTGGAAAGTCCCTCGAAGAGGGACTTTCCAG-(CH2)3NHR (hairpin decoy NF-κB-ODN) and RHN(CH2)6-TGCAGTCACTACGCGAAGCGTAGTGACTGCA-(CH2)3NHR (hairpin scrambled decoy NF-κB-ODN) where R is either H or biotin. The synthesis of decoy oligonucleotides with R = H has been published elsewhere . For biotin addition, 7-10 nanomoles of the oligodeoxynucleotide bearing 3'- and 5'-aminoalkyl linkers were dissolved in 20 μL of 0.1 M NaHCO3. EZ-Link NHS-biotin (Pierce, Rockford, USA) (10 μL of a 65 mM solution in dimethyl sulfoxide) was added, and the mixture was incubated at room temperature for 6-16 h in the dark. Then 25 μL of water were added, and the modified oligodeoxynucleotide was separated from the excess of hydrolyzed reagent by two consecutive separations on Micro Bio-Spin 6 columns following the manufacturer's recommendations. After the second spin, the biotinylated oligodeoxynucleotide was precipitated with ethanol-sodium acetate. In control experiments the previously published decoy NF-κB-ODN  was used. In some cases FITC-labeled or biotinylated decoy ODNs were obtained from Sigma-Aldrich. Note that the oligonucleotides used for cell death induction, pull-down assays and whole-cell pull-down assays were similar and could be used interchangeably, except that for pull-down biotinylated oligonucleotides had to be used.
Preparation of liposomes
Liposomes were formulated using a cationic lipid (3β-[N-(N',N',N'-triethylaminopropane)-carbamoyl] cholesterol) iodide (TEAPC-Chol) and neutral colipid dioleoyl phosphatidylethanolamine (DOPE), as previously described . The concentration of cationic lipid was monitored by UV spectroscopy at 226 nm and the value was used to calculate the charge ratio assuming one positive charge for each cationic lipid molecule.
Gel electrophoresis, western blotting
Cells were washed in PBS, lysed in sample buffer (50 mM Tris-HCl pH 6.8 (Bio-Rad, Marnes-la-Coquette, France), 2% sodium dodecyl sulfate (SDS) (Sigma-Aldrich), 20% glycerol (Prolabo, Fontenay-sous-Bois, France), 1 mM sodium vanadate (Na3VO4, Labosi, Elancourt, France), 1 mM dithiothreitritol (DTT) (Merck, Fontenay Sous Bois, France) and 0.01% bromophenol blue (Sigma-Aldrich), sonicated and stored at -70°C. Proteins (50 μg) were separated on SDS-PAGE (10%) and transferred onto nitrocellulose membranes; membranes blocked with 5% dry skimmed milk in TBS were incubated with antibody overnight at 4°C. Anti-phosphotyrosine 705-STAT3 (1/1,000), anti-STAT3 (1/1,000), anti-NF-κB p50 (1/1,000), anti-NF-κB p65 (1/1,000), anti-STAT1 (1/1,000), and anti-OCT1 (1/1,000) were from Cell Signaling, anti-karyopherin/importin α (1:400) was from Santa Cruz (Tebu-bio, Le Perray en Yvelines, France). Blots were washed in TBS-T, incubated with peroxidase-coupled goat anti-mouse (Santa Cruz, Tebu-bio) or goat anti-rabbit (Upstate, Ozyme) secondary antibody (1/20,000) washed in TBS-T and revealed by chemiluminescence (LumiGLO reagent and peroxide; Cell Signaling) and autoradiography (X-Omat R film; Kodak). When necessary, membranes were stripped with Blot Restore Kit (Chemicon International) and reprobed with anti-actin antibody (Cell Signaling). Prestained molecular weight standards (Fermentas, Saint-Rémy-lès-Chevreuse, France) were used. For the quantification analysis, the bands from at least three separate experiments were scanned using a Chemidoc apparatus (Biorad) and quantification performed using the Quantity One software (Biorad). P-values were calculated using a t test.
The TaqMan® Gene Expression Cells-to-CT™ kit (Applied Biosystems, Courtaboeuf, France) was used to extract total RNA and to perform reverse transcription and gene amplification. An Applied Biosystems Custom TaqMan Gene Expression Assay was used; the sequences were chosen to cover exons 5 and 6 to avoid detecting genomic DNA: sense primer: 5'-ccatcttcatcacactcttcctgtt, antisense primer: 5'-accaccgaggagaagatcca, 5'-FAM probe: 5-ctacagtgccaccgtcacc. For the TaqMan Gene Expression Assay (Applied Biosystems) ref. Hs00941525_g1 was used. For cyclophilin A (PPIA), used as a reference, the TaqMan Gene Expression Assay, ref. Hs99999904_m1 was used. All steps were performed following the recommendations of the manufacturer. Relative expression levels of each gene were calculated as previously described.
Cells were grown in 4-well plates to a density of 0.5 106 cells/mL. When the cells reached 50-60% confluence, they were transfected with STAT3-decoy ODN or the hairpin control decoy ODN (2 μg corresponding to 400 nM) in 150 μL of DMEM medium (without SVF) combined with the liposomes (2 μg of cationic lipid). After 6 h at 37°C in a humidified 5% CO2 incubator, the cells were placed in fresh serum-containing medium. Expression was analyzed after 48 h. In other cases, transfection was performed using polyethyleneimine (PEI, Sigma-Aldrich), with an ODN-to-polyethyleneimine ratio of 1:1.
Flow cytometry, cell viability, immunocytochemistry
To measure cell death, cells were resuspended in annexin V-binding buffer, incubated with 5 μL of propidium iodide (BD Pharmingen, Morangis, France) and subjected to flow cytometry analysis, using a BD FACS Canto II Flow Cytometer. Cell viability was also assessed using the trypan-blue exclusion method with a V-cell counter (Beckmann, Villepinte, France).
For immunocytochemistry, cells were grown in 8-well plates (lab-tek, Nunc, Rochester, USA) to a density of 0.5 106 cells/mL. At 50-60% confluence, cells were transfected with the FITC-labeled STAT3-decoy ODN or the FITC-labeled mutated STAT3-decoy ODN. After 48 h the cells were washed in NaCl-phosphate buffer, fixed in 3.7% formaldehyde for 15 mn, permeabilized in 0.1% Triton X-100 for 15 mn and blocked in 5% FCS, 0.1% Tween in NaCl-phosphate buffer for 1 h. Cells were stained with anti-STAT3 antibody (Cell Signaling) (dilution: 1:100) or anti- phosphotyrosine 705-STAT3 antibody (Cell Signaling) (1:100) for 2 h and Alexa Fluor 546-labeled secondary antibody (Invitrogen) (1:200) for 90 mn. After counterstaining with 4', 6'- diamidino-2-phenylindole (DAPI) coverslips were mounted onto glass slides with Vectashield (Vectorlabs, Clinisciences, Montrouge, France). Fluorescence images were acquired using a Zeiss Axioplan2 Deconvolution microscope (Carl Zeiss, Le Pecq, France) and analyzed with Metafer4 (Metasystems, Altlussheim, Germany).
Nuclear protein extracts were obtained as follows: 20 million cells were resuspended in lysis buffer (20 mM Hepes, pH 7.4, 1 mM MgCl2, 10 mM KCl, 0.3% NP40, 0.5 mM dithiothreitol, 0.1 mM EDTA, protease inhibitors: Compete™, Boerhinger) at 4°C for 20 min. The lysates were centrifuged at 14 000 × g for 5 min at 4°C, and the supernatants containing the cytoplasmic proteins were discarded. The pellets were resuspended in the cell lysis buffer adjusted with 20% glycerol and 0.35 M NaCl for 30 min at 4°C. After centrifugation at 14 000 × g for 5 min at 4°C, the supernatants were stored at -80°C. For pull-down assays, 100-200 μg of nuclear protein extracts were incubated for 30 min at 4°C in binding buffer (1% NP40, 50 mM Hepes, pH 7.6, 140 mM NaCl) containing salmon sperm DNA (1 μg/assay) and 1 μg of the biotinylated hairpin decoy ODN or the mutated decoy ODN. The complexes were captured by incubation with 50 μl of avidin-Sepharose beads (neutravidin, Pierce) for 2 h at 4°C. For in-cell decoy ODN pull-down assays, the cells were first transfected with STAT3-decoy ODN or its mutated equivalent, as described under oligonucleotide transfection (see above), and then processed as above by cell lysis and recovery on avidin-Sepharose beads. After extensive washing with binding buffer, complexes were separated on SDS-polyacrylamide (8%) gel, and subjected to immunoblotting using an anti-STAT3 antibody (Cell Signaling). Results were analyzed by chemiluminescence (LumiGLO, Cell Signaling) and autoradiography (X-Omat R, Kodak).
For antibody pull-down assays, 20 million cells were lyzed and resuspended in lysis buffer (20 mM Hepes, pH 7.4, 1 mM MgCl2, 10 mM KCl, 1% NP40, 0.5 mM dithiothreitol, 0.1 mM EDTA, 1 mM orthovanadate, protease inhibitors; Compete™, Boerhinger) at 4°C for 5 min. The lysates were centrifuged at 14 000 × g for 5 min at 4°C, and the supernatants containing the cytoplasmic proteins were either used immediately or stored at -80°C. For the immunoprecipitations, 200 to 400 μg protein was supplemented with albumin-saturated protein G-agarose (Boehringer); after centrifugation (8000 × g, 5 min), the pellet was discarded and the supernatant conserved. Antibody was added (STAT3: 1:100; karyopherin, Santa-Cruz: 1:40) and incubation continued overnight at 4°C. Samples were then supplemented with albumin-saturated protein G-agarose and incubated for 1 h 30 m. The agarose beads were washed three times with TBS and once with TBS-T, and resuspended in SDS-sample buffer. Gel separation and western blotting were performed as described above.