Flow cytometry Aliquots of cells (26105 per well) were seeded in fully supplemented appropriate medium into 24-well plates. 24 h later, the cells were washed three times with PBS, and 500 ml of appropriate medium containing 1% penicillin/streptomycin and 10% glutamine but no FCS was added. For transduction assays, different vector preparations were added to the medium at various MOI, with or without incubation with FX at different concentra- tions. After 2 h, the cells were extensively washed with PBS prewarmed at 37uC, then 1 ml of fresh fully supplemented medium was added and cells further incubated at 37uC for an additional 22 h. Cells were resuspended in PBS and analyzed by flow cytometry, using a FACSCantoTM II cytometer (Becton Dickinson Biosciences). 20,000 events were acquired for each sample and the results were analyzed using the DIVA 6 software (Becton Dickinson). Surface plasmon resonance (SPR) assays The source of naturally hypersulfated heparin (HS), sodium salt, was the porcine intestinal mucosa (Sigma). HS calibrated to 9 kDa was biotinylated and immobilized on a CM4 BIAcore sensorchip (GE Healthcare, Saclay, France), using a BIAcore3000 (GE Healthcare), as previously described [48,49]. Two flowcells were prepared by sequential injections of EDC/NHS, streptavidin, and ethanolamine. One of these flowcells served as negative control, while biotinylated heparin was injected on the other one, to get an immobilization level of 80–90 response units (RU). All SPR experiments were performed using HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) supplemented by 3 mM CaCl2, at a flow rate of 5 ml/min. Interaction assays involved injections of different amounts of protein or virus over the heparin-coated and negative control surfaces, followed by a 3-min dissociation time with buffer. At the end of each cycle, the heparin surface was regenerated by injections of 2 M NaCl (2 min). Sensorgrams shown corresponded to on-line subtraction of the negative control to the heparin surface signal. Preparation of plasma membrane-shedded microvesicles (MVs) and MVB-released exosomes (EXOs) (i) Source of MVs and EXOs. Confluent CHO-K1 cells were transduced with HAdV5wt or HAdV5F35 at MOI 5,000, with or without FX, and harvested at 2 h and 24 h pi. The cell culture medium was harvested and floating cells were pelleted by centrifugation at 2,0006 g for 10 min. The supernatant was further clarified from cell debris by centrifugation for 2 min at 13,0006 g and 4uC. This final supernatant (S0) was the source of plasma membrane-derived MVs and EXOs, prepared as described in [96]. (ii) Isolation of MV. Fraction S0 was centrifuged for 2 h at 30,0006 g and 4uC, and pellet P1 was saved: it mainly contained MVs. (iii) Isolation of EXOs. Supernatant S1 was further centrifuged at 100,0006 g and 4uC for 2 h, giving supernatant S2, which was discarded, and pellet P2, which contained EXOs. Pellets were resupended in PBS and subjected to DNA extraction, using QIAamp DNA Blood Mini kit (Quiagen). Quantification of AdV vector genomes by real-time PCR and real-time RT-PCR HAdV5 vectors which were possibly carried over with MVs were detected and quantitated by real-time PCR, using HAdV5 or HAdV35 specific primers and probe selected from the fiber gene. Extraction of DNA was carried out using the QIAamp DNA Cellular Trafficking of FX-Ad5fiber35 Complex Blood Mini Kit (Quiagen) and real time PCR reactions were carried out using the LightCycler DNA Master SYBR Green I kit (Roche) and the LightCycler instrument (Roche). For the serotype 5 fiber gene, the primers used were: sense 59-GCTACAGTTT- CAGTTTTGGCTG-39 and antisense 59-GTTGTGGCCAGAC- CAGTCCC-39 ; for the serotype 35 fiber gene, the primers used were : sense 59- TGGCTTCACACAAAGCCCAGACG-39 and antisense 59- ACACGTAGCCATTAACAAGCCCTCC-39. As internal control, ß-actin gene was amplified using the following primers, sense 59-GCTGTGTTCTTGCACTCCTTG-39 and antisense 59- CGCACGATTTCCCTCTCAGC-39. Proteins FX, activated FX (FXa) which lacked the RGD motif contained in the activation peptide [97], and the truncated form FXGL (Gla domainless) were all purchased from CRYOPREP (Montpellier, France). Proteolytically inactive human FX, blocked with Dansyl-EGR (FX-DEGR), was also purchased from CRYOPREP. HAdV5wt hexon, penton (penton base + fiber) and fiber proteins were isolated from lysates of HAdV5wt- infected 293 cells [85]. HAdV5wt penton base and penton base RGD-340-EGD mutant, were recombinant proteins isolated from recombinant baculovirus-infected cells [98,99]. Viral proteins were purified according to a conventional protocol adapted to fast protein liquid chromatography [100–103]. Protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting, as previously described [85]. Adenovirus type 3 penton dodecahedron (Pt-Dd) purification, labeling and intracellular trafficking HAdV3 Pt-Dd was produced in recombinant baculovirus- infected insect cells coexpressing HAdV3 penton base and fibre proteins, as previoulsy described [40,77–79]. HAdV3 Pt-Dd was purified by sucrose gradient density, and dialysed against HEPES- NaCl buffer (20 mM HEPES, pH 7.4, 150 mM NaCl). Fluores- cent-labeled Pt-Dd was obtained by incubation of a Pt-Dd protein solution at 1 mg/ml with Cy5-monoNHS-Ester at 1 mM (GE Healthcare, PA15101) for 2 h at room temperature, followed by extensive dialysis against PBS to remove unbound dye. Cy5- labeled Pt-Dd (10 mg/ml) was incubated at 37uC for 10 min to 2 h with CHO-CD46 cells transduced one day before with recombi- nant baculovirus suspension expressing the Lamp1 fluorescent marker of the late endosome/lysosome compartment (CellLightTM Lysosomes-RFP *BacMam 2.0*; Invitrogen), as described above. After removal of Cy5-labeled Pt-Dd and rinsing the cell monolayer with prewarmed medium, live imaging was performed using the LSM 510 Meta inverted confocal laser scanning microscope (Carl Zeiss, IAB) as above, with the RFP and Cy5 filters. Electron microscopy (EM) Specimens were processed for EM and observed as previously described [104,105]. In brief, cells were harvested at 2 h pi, pelleted, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.5, post-fixed with osmium tetroxide (2% in H20) and treated with 0.5% tannic acid solution in H20. The specimens were dehydrated and embedded in Epon (Epon-812; Fulham, Latham, NY). Ultrathin sections were stained with 2.6% alkaline lead citrate and 0.5% uranyl acetate in 50% ethanol, and post- stained with 0.5% uranyl acetate solution in H2O. Grids were examined under a Jeol JEM-1400 electron microscope, equipped with an ORIUSTM digitalized camera (Gatan France, 78113- Grandchamp). For statistical EM analyses, a minimum of 50 grid PLoS ONE | www.plosone.org 16 May 2011 | Volume 6 | Issue 5 | e18205