Figure 2
Infectivity of various cell lines with the adenovirus displaying the selected peptide. The 1 × 104 cells were infected with adenoviruses at multiplicity of infection (MOI) of 30 in 96-well plates, and 24 h later the enhanced green fluorescent protein (EGFP)-positive cells in the wells were counted under fluorescence microscope. The 1 × 105 cells were infected with adenoviruses at MOI of 30 in 6-well plates, and 24 h later the flow cytometry was carried out using FACScan systems (Becton Dickinson, Franklin Lakes, NJ, USA) to analyze the frequency of EGFP-positive cells. (a) Infectivity of AsPC-1 cells with adenovirus vectors ablated for coxsackievirus and adenovirus receptor (CAR) binding. Left: representative photographs of EGFP-expressing cells ( × 100 original magnification). Middle: number of EGFP-positive cells counted per well in 96-well plates under microscope. The assays (carried out in eight wells) were repeated a minimum of two times and mean±s.d. are presented. Right: frequency of EGFP-positive cells by flow cytometry. (b) Relative number of EGFP-positive cells after infection of adenovirus vectors ablated for CAR binding in various cell lines. Left: relative number of EGFP-positive cells (the number of EGFP-positive cells infected with AdΔCAR-SYE/AdΔCAR) counted under fluorescence microscope. The statistical differences between the number of EGFP-positive cells infected with AdΔCAR-SYE and with AdΔCAR were as follows: BxPC-3; P=0.0015, AsPC-1; P=0.000013, Panc-1; P=0.000080, MIAPaCa-2; P=0.000013, PSN-1; P=0.41, U138MG; P=0.41, PC3; P=0.11, human umbilical vascular endotherial cells (HUVEC); P=0.20, human pancreatic epithelial cells (HPE); P=0.25. Right: relative frequency of EGFP-positive cells (the frequency of EGFP-positive cells infected with AdΔCAR-SYE or Ad-EGFP/that with AdΔCAR) by flow cytometry. The statistical differences between the frequency of EGFP-positive cells infected with AdΔCAR-SYE and that with AdΔCAR were as follows: BxPC-3; P=0.00000053, AsPC-1; P=0.000039, Panc-1; P=0.0000026, MIAPaCa-2; P=0.00029, PSN-1; P=0.079, U138MG; P=0.085, PC3; P=0.21, HUVEC; P=0.21, HPE; P=0.15.
