FIG. 3.

MBCD interfered with BKV infection. HRPTEC were preincubated with MBCD (1.25, 2.5, and 5 mM) for 1 h prior to coincubation with BKV (MOI, 0.5 FFU/cell). After 72 h, medium was removed and cells were washed three times with REBM with 0.5% FBS and incubated for another 48 h with fresh medium containing MBCD. (A) After incubation, cells were fixed and analyzed by IF (magnification, ×10). T-Ag-positive cells were counted as BKV-infected cells, and the percentage was calculated against total cells. In the experiments whose results are shown in Fig. 3 and 4, untreated HRPTEC and HRPTEC incubated with BKV only were used as negative and positive controls. At least 500 cells were counted from three independent coverslips, and means and SE were calculated from the results of two independent experiments. *, P < 0.02; **, P < 0.01; ***, P < 0.005. (B) After incubation, cells were harvested and analyzed by WB. Relative levels of T-Ag expression were detected by using an Odyssey system to measure the intensities and depicted as graph bars. The intensity of T-Ag expression was corrected by the intensity of GAPDH as the loading control. Means and SE were calculated from the results of two independent experiments. Control, HRPTEC were not incubated with either BKV or MBCD; *, P < 0.05. (C) HRPTEC were preincubated with 5 mM of MBCD for 1 h prior to an 8-h coincubation with labeled BKV (MOI, 5 FFU/cell) and transferrin, (10.0 μg/ml). After cells were fixed, Alexa Fluor 488-labeled BKV (green) and Alexa Fluor 633-conjugated transferrin (red) were observed by confocal microscope using a 63× lens objective.