Production of recombinant protein and chemical compounds
Recombinant 16 K hPRL was produced and purified from E. Coli as previously described . The purity of the recombinant protein exceeded 95% (as estimated by Coomassie blue staining) and the endotoxin level was found to be 0.5 pg/ng recombinant proteins, as quantified with the "Rapid Endo Test" from the European Endotoxin Testing Service (Lonza, Verviers, Belgium). BAY 1170-82 was purchased from Calbiochem (La Jolla, CA).
ABAE (Adult Bovine Aortic Endothelial) cells were isolated as previously described . The cells were grown in low-glucose DMEM containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. Recombinant bFGF (Promega) was added (1 ng/ml) to the culture every other day. Confluent cells corresponding to passages 8 to 13 were used in the experiment. HMVEC (Human Microvascular Endothelial cell) cultures were maintained in EBM2 medium (Lonza, Walkersville, Walkersville, USA) containing 0.1% hEGF, 0.04% hydrocortisone (kit EGM-2 SingleQuots, Cambrex Bio Science Walkersville, Walkersville, USA), 10% FBS, and 100 U/ml penicillin/streptomycin. HCT116 cells (human colorectal carcinoma cells) were grown in McCoy's 5a medium containing 10% FBS and 100 U/ml penicillin/streptomycin. HEK 293 (Human Embryonic Kidney) cells and adenovirus-E1-transformed HEK 293 cells (BD Biosciences, San Diego, CA) were grown in DMEM supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, 100 U/ml penicillin/streptomycin, and 2.5 μg/ml fugisone.
16 K-Ad is a defective recombinant E1-E3-deleted adenovirus vector encoding a secreted peptide consisting of the first 139 amino acids of PRL. This adenovirus vector was constructed as described in  with the help of the Adeno-X expression system (BD Biosciences, Erembodegem, Belgium). Briefly, the 16 K hPRL complementary DNA was cloned into a pShuttle vector in an expression cassette, which was then inserted into the Adeno-X viral DNA. Recombinant adenoviruses were constructed and amplified in HEK 293 cells. The BD Adeno-X Virus Purification kit (BD Biosciences, Erembodegem, Belgium) and the Adeno-X Rapid Titer Kit (BD Biosciences, Erembodegem, Belgium) were used to perform purification and titration, respectively, of the recombinant adenoviruses (according to the manufacturer's instructions). Null-Ad is a control adenovirus carrying an empty expression cassette.
Adult female NMRI nude mice (6-8 weeks of age) purchased from Janvier Breeding (Elevage Janvier, France) were used for tumor growth experiments. The animal experiment protocol used was approved by the Institutional Ethics Committee of the University of Liege.
Mouse xenograft tumor model
Subconfluent HCT116 cells were trypsinized, washed, and resuspended in PBS. Cell suspension (3.106 cells in 50 μl PBS) was injected s.c. into the right flank of NMRI nude mice 2 weeks before the first adenovirus administration. Sixteen mice were used and randomly divided into two groups of 8 mice. Mice received four intratumoral injections of 5.108 pfu 16 K-Ad or Null-Ad (control) starting when the HCT116 tumors reached 150 mm3. These injections were repeated every 2 days. Ten days after the first adenoviral vector injection, the mice were euthanized and their tumors harvested. Tumor growth was assessed by measuring the length and width of each tumor every 2 or 3 days and calculating tumor volume by means of the formula: length × width2 × 0.5 .
Small interfering RNA (siRNA) duplexes were obtained from Integrated DNA Technologies (Integrated DNA Technologies, Coralville, USA), two targeting SPRY1 and one negative control. Cells were transfected by the CaPO4 method. Briefly, 90,000 ABAE cells were seeded into a 6-well plate and allowed to adhere overnight. One hour before transfection, the medium was replaced with fresh medium without antibiotics. SiRNA-CaCl2 complexes were made by first combining siRNA with 10 μl of 2.5 M CaCl2. One hundred microliters of HSBP (280 mM NaCl, 1.9 mM Na2HPO4; 12 mM glucose, 10 mM KCl; 50 mM Hepes, pH 7.05) were added and the mix was incubated for one minute at room temperature. Next the mix was added dropwise to the cells followed by an incubation period of 16 h. Cells were then collected and seeded for further tests.
Quantitative real time PCR (qRT-PCR) analysis
Total RNA was extracted with the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Synthesis of cDNA was performed from 1 μg total RNA, which was reverse transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Clinical Laboratories, Indianapolis, IN) according to the manufacturer's instructions. The resulting cDNA was used for quantitative real-time PCR with the one-step 2× Mastermix (Diagenode, Liège, Belgium) containing SYBR green. Thermal cycling was performed on an Applied Biosystem 7000 detection system (Applied Biosystems, Foster City, CA). No-template controls were run for all reactions, and random RNA preparations were also subjected to sham reverse transcription to check for the absence of genomic DNA amplification. The relative transcript level of each gene was obtained by the 2-ΔΔCt method  and normalized with respect to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (in vitro assays) or cyclophilin A (PPIA) (mouse assays). Primers were designed with the Primer Express software and selected so as to span exon-exon junctions to avoid detection of genomic DNA (see Additional file 3 - List of primers used in quantitative RT-PCR). In order to verify species specificity of the PCR, PCR combining human or mouse cDNAs with human or mouse primers have been performed on cloned cDNAs for PPIA or Sprouty obtained form the German Resource Center for Genome Research (RZPD, IMAGENES, Germany). For analysis by end-point PCR, the final products of the qRT-PCR obtained after 40 cycles of PCR was loaded on agarose gel for electrophoresis.
Preparation of cell extracts
Cells were washed twice with cold PBS and scraped into lysis buffer [25 mM HEPES (pH 7.9), 150 mM NaCl, 0.5% Triton, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride] on ice. Insoluble cell debris was removed by centrifugation at 10000 × g for 15 min. Aliquots of protein-containing supernatant were stored at -80°C. Protein concentrations were determined by the Bradford method, with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA).
Western blot analysis
Soluble cell lysate (30 μg) was resolved by SDS-PAGE (12%) and transferred to a polyvinylidene fluoride membrane (Milipore Corp., Bedford, MA). Blots were blocked overnight with 8% milk in Tris-buffered saline with 0.1% Tween 20 and probed for 1 h (or 2 h at 37°C for anti-SPRY1 antibody) with primary antibodies: anti-Prolactin A602 (home-made), anti-SPRY1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 Map Kinase (Thr202/Tyr204) antibody (Cell Signaling Technology, Beverly, MA), anti-MAP Kinase 1/2 (Millipore, Billerica, MA), polyclonal rabbit anti-beta-tubulin (Abcam plc, Cambridge CB4 OFW, UK). After three washes with Tris-buffered saline containing 0.1% Tween 20, antigen-antibody complexes were detected with peroxidase-conjugated secondary antibody and an enhanced fluoro-chemiluminescent system (ECL-plus; Amersham Biosciences, Arlington Heights, IL).
ABAE cells were fixed with paraformaldehyde 1% for 30 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. The samples were blocked with 0.2% bovine serum albumin in PBS for 30 min and incubated with rabbit anti-SPRY1 over night at 4°C. This was followed by incubation with a goat anti-rabbit-Cy3 for 30 min. Fluorescence was analyzed with an Olympus fluorescence microscope and a camera linked to the Analysis software (Soft Imaging System GmbH, Münster, Germany).
Caspase-3 activity assay
Control and SPRY1-siRNA-transfected cells were plated in 24-well culture plates at a density of 20,000 cells per well in 500 μl of 10% FCS/DMEM. Caspase-3 activity was measured 48 h post-transfection with the CaspACE Assay System Fluorimetric (Promega Corp., Madison, WI) according to the manufacturer's instructions.
Analysis of cell proliferation
Transfected cells were plated in 96-well culture plates at a density of 5,000 cells per well in 10% FCS/DMEM and allowed to adhere for 6 h. Following this, complete medium was replaced with DMEM free for 24 h. The transfected cells were then incubated in 10% FBS/DMEM or DMEM containing 10 ng/ml bFGF and proliferation was analyzed 24 h later by measuring BrdU incorporation by means of the Cell Proliferation ELISA, BrdU (Colorimetric) (Roche, Clinical Laboratories, Indianapolis, IN)
Capillary network formation on a Matrigel matrix
The ability of SPRY1-siRNA-transfected ABAE cells to form capillary networks was evaluated in a Matrigel™angiogenesis assay. Briefly, 80,000 cells were plated in 24-well plates coated beforehand with 300 μl Matrigel. Control-siRNA- and SPRY1-siRNA-transfected cells were seeded into 200 μl of DMEM or 10% FBS/DMEM for 16 h. In order to visualize vessels under a fluorescence microscope, the cells were incubated with calcein-AM (2 μM) for 20 min. Quantitative analysis of network structures was performed by measuring the number of connections between vessels in the network. Photographs were taken with an Olympus fluorescence microscope and a camera linked to the Analysis software (Soft Imaging System GmbH, Münster, Germany)
Eight-micrometer 24-well Boyden chambers (Transwell; Costar Corp, Cambridge, MA) were used for cell migration assays. Both sides of the membrane were coated overnight with 0.005% gelatin. The lower chamber was filled with 600 μl DMEM containing 1% BSA and 10 ng/ml bFGF. ABAE cells transfected with siRNA duplexes, as described above, were placed in 300 μl of 0.1% BSA/DMEM in the upper chamber and allowed to migrate for 16 h at 37°C. After fixation, cells stained with 4% Giemsa were counted on the lower side of the membrane. Cell counting was performed with an ImageJ http://rsbweb.nih.gov/ij/ macro relying on color thresholding in the RGB color space, followed by connected component labeling with the "Analyze Particles" function with size and circularity criteria. The same set of parameters was used for the experiments, and detection masks were produced and double-checked by visual examination.
Cell adhesion experiments were performed in 96-well plates coated with either vitronectin or fibronectin. Wells were coated with 50 μl vitronectin (10 μg/ml) or fibronectin (10 μg/ml) for 1 h, and then washed twice with PBS. Briefly, 50,000 siRNA-transfected cells were plated on the coated 96-well plates and allowed to adhere for 1 h. The wells were then washed twice with medium to remove non-adherent cells. The cells were fixed and stained with 0.01% crystal violet in methanol, then the wells were washed extensively with water and the dye was solubilized in methanol. Quantification was performed by reading the optical density at 550 nm with a microplate reader (Wallac Victor2; Perkin Elmer, Norwalk, Finland).
Luciferase reporter Assays
NF-κB luciferase reporter assays were performed as previously described . Luciferase activity was normalized using the β-galactosidase activity with the β-gal Reporter Gene Assay Kit (Roche).
Quantification and statistical analysis
Quantification of Western blots was performed using ImageJ software http://rsbweb.nih.gov/ij/. All data are expressed as means ± SD unless stated differently. Analyses for statistical significance (the Mann-Whitney test) were carried out with Prism 4.0 software (GraphPad Software, San Diego, CA, USA). Statistical significance was set at P < 0.05.