Antibodies, oligonucleotides, and reagents
Cell culture reagents were from BiochromKG (Seromed, Germany). Human recombinant PTN was from Peprotech Inc (Rocky Hill, NJ, USA) or was prepared as previously described . Monoclonal antibody against NCL, affinity purified rabbit polyclonal antibody against PTN (used for immunoprecipitations and immunohistochemistry of CAM paraffin sections) and goat polyclonal antibody against RPTPβ/ζ were from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Monoclonal antibody against PTN (used only in human cells) was from Abnova (Taipei City, Taiwan), goat polyclonal antibody against PTN was from R&D Systems (Minneapolis, MN, USA), antibody against phospho-ERK1/2 on Thr202-Tyr204 was from Cell Signalling (Danvers, MA, USA), antibody against ERK1/2 was from Upstate Biotechnology (Lake Placid, NY, USA), antibody against Prox-1 was from Axxora (San Diego, CA, USA), antibodies against β-actin and ανβ3 were from Chemicon (Temecula, CA, USA) and monoclonal antibody against RPTPβ/ζ was from BD Transduction Laboratories (San Diego, CA, USA). Protein A and G agarose beads were purchased from Merck (Whitehouse Station, NJ, USA). DNA oligonucleotide primers for chicken PTN and GAPDH and RNA oligonucleotide primers for NCL were obtained from VBC Biotech Services (Vienna, Austria). Double-stranded negative control siRNA was obtained from Ambion (Austin, TX, USA) and the transfection reagents Jet-PEI and JetSI-ENDO were from Polyplus Transfection (Illkirch, France). Alexa secondary antibodies were from Molecular Probes (Carlsbad, CA, USA). Rabbit polyclonal antibody against NCL (used only in immunofluorescent studies), human IgG and all secondary horseradish peroxidase-conjugated antibodies were purchased from Sigma (St. Louis, MO, USA). Draq5 was from Biostatus Limited (Leicestershire, United Kingdom). The 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, Wang resin and peptide reagents were purchased from CBL (Patras, Greece), Bachem (Bubendorf, Switzerland) and Novabiochem (Läufelfingen, Switzerland). All other reagents, unless mentioned below, were purchased from Sigma or Applichem (Darmstadt, Germany).
The in vivo chicken embryo CAM angiogenesis assay was used, as previously described . Leghorn fertilized eggs (Pindos, Ioannina, Greece) were incubated for 4 days at 37°C, when a window was opened on the egg shell, exposing the chorioallantoic membrane. The window was covered with tape and the eggs were returned to the incubator. Different amounts of pCDNA3.1 alone or pCDNA3.1 carrying full length cDNA for PTN in antisense orientation (AS-PTN) , were diluted in a final volume of 50 μl of phosphate buffered saline pH 7.4 (PBS) containing jet-PEI (N/P = 5 ratio) and were applied at the 9th day of embryo development on an area of 1 cm2 of the CAM, restricted by a plastic ring. Forty eight hours after treatment and subsequent incubation at 37°C, CAMs were fixed in situ, excised from the eggs, placed on slides and left to air-dry. Pictures were taken through a stereoscope equipped with a digital camera and the total length of the vessels was measured, as previously described . Assays were carried out three times and each experiment contained 10-20 eggs per data point.
For the biochemical studies, plasmids were applied on the CAM as described above, and after 24 h of incubation at 37°C, the CAMs were excised from the eggs, cut in pieces, washed three times in PBS and stored at -80°C until used . Assays were carried out three times and each experiment contained 5-10 eggs per data point.
Haematoxylin-eosin staining and immunohistochemistry of CAM paraffin sections
Tissues from various developmental stages were excised from the eggs, washed in PBS, fixed in saline-buffered formalin and embedded in paraffin. Sections were cut at 5 μm thickness and placed on positively charged glass slides. After rehydration, the tissue sections were stained with standard haematoxylin-eosin staining or processed for immunohistochemistry. In the latter case, endogenous peroxidase was blocked with 3% H2O2 for 30 min in a dark chamber at room temperature. Tissue sections were then incubated with blocking agent (Kwik Kits, Immunon Immunohistochemicals, Lipshaw, USA) for 15 min at room temperature to prevent non specific binding of antibodies, followed by incubation with 5 μg/ml of affinity purified rabbit anti-PTN IgG in Tris-buffered saline (TBS), pH 7.4, with 0.05% Tween (TBS-T) containing 2% bovine serum albumin (BSA) for 1 h at 37°C. After 3 washes of 2 min each in TBS-T, a second 30 min incubation at room temperature, using a horseradish peroxidase conjugated goat anti-rabbit IgG was performed at a dilution of 1:5,000 in TBS-T containing 2% BSA. After three washes of 2 min each in TBS, detection of PTN was performed by DAB staining. Sections were mounted in mounting fluid, viewed in a Zeiss microscope and photographed using a digital camera .
Human umbilical vein endothelial cells (HUVEC) used in the present study were isolated from human umbilical cords and cultured as previously described . HUVEC were grown as monolayers in medium M199 that was supplemented with 15% fetal bovine serum (FBS), 150 μg/ml endothelial cell growth supplement, 5 U/ml heparin sodium, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin and 2.5 μg/ml amphotericin B and used at passages 2-3. U87MG cells (ATCC) were grown routinely in Dulbecco's modified Eagle medium (DMEM)/Ham's F12 medium supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin, and 2.5 μg/ml amphotericin B. Cultures were maintained at 37°C, 5% CO2, and 100% humidity. When cells reached 70-80% confluence, they were lysed for immunoprecipitation experiments or fixed for immunofluorescent studies.
Migration assays were performed as described previously [3, 5] in 24-well microchemotaxis chambers (Corning, Inc., Lowell, MA, USA) using uncoated polycarbonate membranes with 8-μm pores. Serum-starved cells were harvested and resuspended at a concentration of 105 cells/0.1 ml in serum-free medium containing 0.25% BSA. The bottom chamber was filled with 0.6 ml of serum-free medium containing 0.25% BSA and the tested substances. The upper chamber was loaded with 0.1 ml of serum-free medium containing 105 cells and incubated for 4 h at 37°C. After completion of the incubation, the filters were fixed and stained with 0.33% toluidine blue solution. The cells that migrated through the filter were quantified by counting the entire area of each filter, using a grid and an Optech microscope at X20 (Optech Microscope Services Ltd., Thame, UK).
Western blot analysis
CAMs from various developmental stages or after treatment with the plasmids were homogenized using a glass-glass homogenizer in 20 mM Hepes, pH 7.4, containing 2 M NaCl supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA and 1 μg/ml aprotinin. The homogenate was centrifuged at 10,000 x g for 20 min at 4°C. Equal amounts (100 μg) of total protein from CAM lysates or the immunoprecipitated samples, as described below, were analysed by SDS-PAGE and transferred to Immobilon P membranes. Blocking was performed by incubating the membranes with TBS-T containing 3% BSA in the case of PTN, RPTPβ/ζ, PROX-1, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) and 5% non-fat dry milk in the case of NCL and actin. Membranes were incubated with primary antibodies for 16 h at 4°C under continuous agitation, washed 3 times with TBS-T, and incubated with secondary antibodies for 1 h at room temperature. Membranes were finally washed and detection of immunoreactive bands was performed using the ECL detection kit (Pierce), according to the manufacturer's instructions. Blots for PTN, NCL and PROX-1, where appropriate, were stripped and subjected to subsequent Western blotting for actin. Blots for pERK1/2 were stripped and subjected to subsequent Western blotting for total ERK1/2. The protein amounts that corresponded to each immunoreactive band were quantified from digital images of gels, using the ImagePC image analysis software (Scion Corporation, Frederick, MD) [5, 12].
CAMs or cells were homogenised or lysed, respectively, in PBS containing 1% Triton X-100, 0.1% SDS, 20 nM sodium orthovanadate, 1 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride and 5 mM EDTA. Homogenates or lysates were centrifuged at 20,000 g for 30 min at 4°C. Three mg of total protein were transferred to new eppendorf tubes and incubated with primary antibodies for 16 h at 4°C under continuous agitation. Protein A- and protein G-agarose beads were added and samples were further incubated for 2 h at 4°C. Beads and bound proteins were collected by centrifugation and washed twice with ice-cold PBS . The pellet was resuspended in 50 μl SDS loading buffer, heated to 95-100°C for 5 min, centrifuged and analyzed by Western blot analysis as described above.
Reverse transcriptase-polymerase chain reaction (RT-PCR) for PTN
Total RNA was extracted from CAMs of various developmental stages using the Nucleospin RNA II kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. Primers used for the detection of PTN mRNA were designed according to the chicken sequence (Accession number BI394859) and were: 5'-AGA GAA ACC AGA GAA AAA GG-3' (sense) and 5'-CAG TCA GCA TTA TGA AGA GC-3' (antisense), yielding a product of 288 bp. The reporter gene was the chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the primers used were: 5'-ACG GAT TTG GCC GTA TTG GC-3' (sense) and 5'-GCA GGA TGC GAA ACT GAG CG-3' (antisense) . The RT-PCR reactions for PTN and GAPDH were performed in a single step, using the Access RT-PCR system (Promega) under the following conditions: The reverse transcriptase reaction was performed by AMV-RT for 1 h at 48°C. After an initial denaturation step for 2 min at 94°C, 30 cycles of amplification (94°C for 1 min, 57°C for 40 sec and 68°C for 1.5 min for GAPDH and 94°C for 1 min, 55°C for 40 sec and 68°C for 1.5 min for PTN) were performed and ended with a final DNA synthesis step at 68°C for 7 min. In all cases, PCRs were not in the saturating phase (data not shown). DNA contamination was excluded by performing PCR reactions in the absence of the reverse transcription step. The RT-PCR products were subjected to electrophoresis on 2% agarose gels containing 0.5 μg/ml ethidium bromide and photographed using a digital camera. The bands were quantified (area and intensity) using Image PC image analysis software and the ratios PTN/GAPDH of electrophoretic band values represent the relative expression of ptn gene at different days of embryo development.
The short interfering RNA against NCL was sense: 5'-GGAAGGUCAGCAGUCUUCCAUGAGA-3' and antisense: 5'-UCUCAUGGAAGACUGCUGACCUUCC-3' . HUVEC were grown to a confluence of 50% in medium without antibiotics. Transfection was performed in serum-containing medium for 4 h using annealed RNA for NCL at the concentration of 50 nM and jetSI-ENDO as transfection reagent. Cells were incubated for another 24 h in serum-containing medium and lysed in order to evaluate transfection efficiency by Western blot analysis, or fixed for immunofluorescent studies. Double-stranded negative control siRNA from Ambion (catalogue # AM4635) was used in all assays.
For immunofluorescent studies, cells were fixed by 4% paraformaldehyde for 10 min. After being washed 3 times with PBS, the cells were blocked with PBS containing 3% BSA and 10% FBS for 1 h at room temperature. The cells were stained with primary antibodies against NCL (1:1,000) and PTN (1:500). Nuclei were stained with Draq5 (final concentration 3.3 μM). Fluorescent secondary antibodies were used at the concentration of 1:500, and the cells were mounted with Mowiol 4-88 (Calbiochem, San Diego, CA, USA) and visualized at 21°C with Leica SP5 (X63 objective with a numerical aperture of 1.4; Leica Microsystems, Wetzlar, Germany) confocal microscope.
Subcellular fractions of U87MG cells comprising cytosolic, nuclear and cell membrane extracts were prepared as follows : Cell monolayers in 100-mm plates were washed extensively with PBS before being scraped and pelleted. Washed cells (30 × 109) were then disrupted in a hypotonic solution (10 mM Hepes, pH 6.9, 10 mM KCl, 2 mM MgCl2, 1000 units/ml aprotinin, 0.1 mM PMSF) on ice. Nuclei were pelleted at 400 g for 5 min and washed twice in PBS before extraction in the lysis buffer (10 mM Tris-HCl, pH 7.6, 400 mM NaCl, 1 mM EDTA, 1000 units/ml aprotinin, 0.1 mM PMSF, and 1% Triton X-100). The lysate was centrifuged at 12,000 g for 10 min and the supernatant was referred to as the nuclear fraction. The supernatant obtained after pelleting intact nuclei was further centrifuged at 14,000 g for 30 min and the supernatant corresponding to the cytosolic fraction was recovered, while the pellet was resuspended in lysis buffer containing 150 mM instead of 400 mM NaCl. This latter suspension was centrifuged at 14,000 g for 30 min to separate the cytoskeletal (the pellet) and membrane (supernatant) fractions. Equivalent total protein amounts of each fraction corresponding to nuclei, cytosol, and membrane were immunoprecipitated for PTN and then analyzed by Western blot analysis for PTN or NCL.
Synthesis of the 5(KPR)TASP peptide
5(KPR) TASP peptide, a potent and selective ligand of cell surface nucleolin, was synthesized by the solid phase method using protected Fmoc amino acids and Wang resin as the solid support . In summary, Fmoc-protected amino acids were used with the t-Butyl group as side-chain protecting group for Glu, tert-butuloxycarbony group (t-Boc) and/or 4-methoxytrityl group for Lys, t-Boc group for Trp, trityl group for Cys and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group for Arg. Stepwise synthesis of the TASP core peptide was achieved using diisopropylcarbodiimide/1-hydroxybenzotriazole as coupling reagents. The KPR tripeptide was step by step synthesized on ε-aminogroup of the lysine residues in positions 1,3,6,8 as well on the aminoterminal of the TASP core using HATU/DIPEA in dimethylformamide as coupling agents. After completion of the synthesis, the resin was treated with a trifluoroacetic acid solution (TFA/1,2-ethanedithiol/triethylsilane/water/anisole, 95/1/1/1/2 v/v/v/v/v) in the presence of scavengers to liberate the fully deprotected crude peptide. The released peptide was precipitated upon solvent concentration and addition of cold ether and the final product was purified by gel filtration chromatography on Sephadex G-15 using 20% acetic acid as the eluent. Final purification was achieved by preparative high performance liquid chromatography. The final 5(KPR)TASP peptide was checked for its purity by analytical HPLC on a Lichrosorb RP18 column (C18 solid phase, 7 μm particle size, 250 mm × 8 mm) applying a linear gradient 10%-70% acetonitrile (0.1% TFA) for 35 minutes, and 70%- 100% acetonitrile (0.1% TFA) for 5 minutes (flow rate 1.5 mL/min, UV detection at 220 nm and 254 nm). The final verification of the peptide sequence was achieved by Electron Spray Ionization-Mass Spectrometry.
The significance of variability between the results from various groups was determined by one way ANOVA. Each experiment included triplicate measurements for each condition tested, unless otherwise indicated. All results are expressed as mean ± S.E.M. from at least three independent experiments.