Article Figures & Data
Figures
- FIG. 1.
EM of rAd and eAd. Suspensions of virus were examined by transmission EM using a negative-stain technique. A glow-discharged Pioloform-filmed 400 mesh nickel grid was placed film side down onto a 10-μm drop of viral suspension for 10 min to allow for particle adsorption. The grid was rinsed briefly to remove buffer salts by floating on 2 drops of distilled H2O and stained by transferring it to a drop of 2% aqueous uranyl acetate, pH 3.5 (or 2% potassium phosphotungstate, pH 7.0), for 1 min. The stained grids were air dried and examined at an accelerating voltage of 80 keV in a LEO EM-910 transmission EM (LEO Electron Microscopy, Inc., Thornwood, N.Y.). Magnification, ×160,000. Two populations of virus are apparent here. (A) Empty particles; (B) rAd.
- FIG. 2.
AFM of rAd and eAd. The silicon substrate surface with intact native oxide was prepared by UV cleaning, rinsing with distilled H2O (dH2O), and drying under a filtered-nitrogen stream. Approximately 15 μl of solution of 1011 Ad particles/ml was deposited on the above surface, and viruses were allowed to adsorb for 15 min. Unbound viruses were removed by washing with dH2O and then dried under a nitrogen stream. Imaging was performed in air with a ThermoMicroscopes Explorer (Veeco/TM, Sunnyvale, Calif.) with a 100-μm tripod air scanner calibrated with a NT-MDT calibration grid. Silicon tips (Nanosensors FESP; Digital Instruments, Santa Barbara, Calif.) with spring constant of ∼2 N/m and resonance frequency of ∼70 kHz were used for imaging in intermittent-contact mode. (A) rAd; (B) empty particles.
- FIG. 3.
Competition of eAd with rAd. Ad mixtures with approximately 5 × 105 rAd-GFP particles were incubated with 105 IMR-90 cells at 4°C for 1 h to allow virus attachment in cold DMEM with 2% FCS. The cells were then washed three times in ice-cold PBS to remove unattached virus. DMEM with 10% FCS was then added, and cells were placed at 37°C in 5% CO2. At 24 h cells were examined for GFP expression by fluorescence microscopy with a fluorescein isothiocyanate filter. GFP-expressing cells were counted, and duplicate experiments yielded similar results. IMR-90 cells were demonstrated to express the Ad receptor, CAR, on their surface by immunohistochemistry (data not shown).
- FIG. 4.
Internalization of eAd. For internalization studies IMR-90 cells were grown on eight-well chamber slides (LabTech II) and then infected with Cy3-labeled Ad (red) at approximately 10,000 particles per cell. Cells were fixed at 6 h p.i. with 4% paraformaldehyde in PBS for 15 min. They were then mounted with Vectashield containing DAPI to label nuclei (blue) (Vector Laboratories, Burlingame, Calif.). Internalization was assessed by confocal microscopy with a Zeiss CLSM 210 confocal laser scanning microscope with a UV laser.
Tables
- TABLE 1.
Genes modulated by eAd and rAd at 24 h p.i.
a A ∼ in front of a value indicates that the transcripts in one of the samples did not register as present and therefore the change was calculated against background.
b Red, immune response; blue, stress response; green, transcriptional regulation; purple, RNA processing.
