Effects of LY294002 on BCRP expression and function. (A) Effects of LY294002 on BCRP protein expression. Cells were cultured in the absence or presence of various concentrations of LY294002 for 24 h. After harvesting the cells, protein sample (20 μg) was loaded in each lane and western blot analysis was performed. (B) FACS analysis of BCRP expression on the cell surface. Cells were incubated for 12 h with or without 2 μM of LY294002, respectively. Trypsinized cells (5 x 105–106) were incubated with either biotinylated mouse IgG or anti-human ABCG2, 5D3, washed and incubated with R-phycoerythrin-conjugated streptavidin. Bold lines indicate fluorescence in the presence of 5D3 and dotted lines indicate control fluorescence in the presence of mouse IgG. (C) Effects of LY294002 on cellular topotecan uptake of MCF-7/BCRP cells. Trypsinized cells (5 x 105) were pre-incubated with indicated concentrations of reagents for 60 min. The cells were then incubated with (solid line) or without (dotted line) 40 μM of topotecan for 30 min in the absence or presence of the reagents. Finally, cellular topotecan uptake was measured by flow cytometry. Reproduced by permission from Spandidos Publications. Inc. Imai Y, et al. (19).