Figure 5
Effect of B7-H1 silencing on cell binding and uptake of Ad5FB4 by DA1-3b/d365 cells. (A, B), Ad5FB4-cell binding. Cells were transfected with control siRNA or siRNA against B7-H1 (3 nmol/cell), and maintained in culture for 144 h. They were then incubated with FAM-labeled Ad5FB4 at 104 vp/cell for 90 min at 4°C. (A), The efficiency of B7-H1 silencing was verified by flow cytometry analysis of siRNA-treated cells, using PE-labeled antibody against murine B7-H1. Curves were: a, control siRNA; b, siRNA against B7-H1; c, control irrelevant isotypic antibody. (B), Cell-bound Ad5FB4 vector particles were quantitated by flow cytometry, and results expressed as the percentage of positive, fluorescent cells. (C, D), Cellular uptake and transduction. Cells were transfected with control siRNA or siRNA to B7-H1 (3 nmol/cell) and maintained in culture for 144 h. They were incubated with FAM-labeled Ad5FB4 at 104 vp/cell for 90 min at 4°C, followed by transfer to 37°C for different periods of time, ranging from 5 to 120 min. (C), Cellular internalization of Ad5FB4 particles. Intracellular vector was quantitated by flow cytometry, and results expressed as the percentage of positive, fluorescent cells. The numbers on top of the rightmost series of bars represented the percentage of decrease in the number of positive cells, relative to that number in control siRNA-treated cell samples at time 0 of transfer to 37°C, which was attributed the 100%-value. (D), Ad5FB4-mediated DA1-3b/d365 cell transduction. B7-H1 siRNA-treated cells were taken at 144 h after transfection and incubated with the Ad5FB4 vector for 2 h at 37°C at increasing vector doses, ranging from 5 × 102 to 104 vp/cell. After an additional 72 h at 37°C, ß-gal activity was assayed in cell lysates, and expressed as MFI. The numbers on top of the rightmost series of bars represented the decrease of ß-gal activity (as a percentage of control), relative to the value in control siRNA-treated cells infected with 5 × 102 vp/cell, which was attributed the 100%-value. MOI, multiplicity of infection, expressed as vp/cell.