Quantitation of co-localization of CB-D28k ir with WNV env ir.

<p>(A) Stable transfection. Cell culture mixture of 50% CB-D28k stably transfected NSC34 cells and 50% vector control stably transfected NSC34 cells at 70–80% confluence were infected with 5 MOI and immunostained 48 hr later. The analyzed images were obtained from 5 representative areas of the slides using confocal fluorescent microscope. From those images, using ZEN software, based on the intensity of the staining, WNV-infected and CB-D28K transfected cells were quantified and plotted. The area within the dotted-line box represent cells where some level of co-localization of CB-D28k ir and WNV env ir was detectable. If CB-D28k did not affect viral replication, there would be no statistical difference between 50% of the number inside the box compared with 50% of the number outside the box. Therefore, chi square statistical analysis was performed by comparing the number of cells co-localized inside the box with the number of cells outside the box. (B) Transient transfection. NSC34 cells were transiently transfected with CB-D28k expression plasmid, and seeded at 70% confluence on glass coverslips. The next day the cells were infected with 5 MOI and analyzed by flow cytometry (total of 73 cells analyzed). Chi square statistical analysis was performed by conservatively assuming at an infection of 5 MOI that at least 60% of the CB-D28k ir cells would be infected by WNV, and co-localized with WNV env ir cells, and comparing the numbers inside and outside the box. P≤0.001. (C) Frequency of distribution of CB-D28k ir in stably transfected NSC34 cells. A total of 260 cells in fields of 100% stably transfected cells through three rounds of cloning were individually measured for relative ir and plotted on a frequency distribution histogram. Y-axis is represented as the percent (%) of total frequency.