Effect of ABCG2 silencing on [3H]gefitinib accumulation, efflux, and uptake.
H460 cells were transfected with ABCG2 siRNA (1:1:1 mixture of #HSS114013, #HSS114014 and #HSS114015) or control siRNA (scr) for 48 hours and then analyzed for ABCG2 expression by Western blotting (A) or ABCG2 activity (B). Cells were incubated for 4 hours with 1 μM Hoechst 33342 in the presence or in the absence of Fumetrimorgin C and the relative ABCG2 activity was calculated as the ratio of Hoechst 33342 accumulation per μg of protein between Fumitremorgin C treated cells and untreated cells, and expressed as fold increase. Data are expressed as mean (± SD) of three different experiments (***P < 0.001). In H460 transfected cells, radiolabeled gefitinib accumulation was measured after 4 hours of treatment with 0.1 μM [3H]gefitinib (C), then the medium was replaced with fresh medium and the percentage of efflux was calculated after 5, 10, 20 and 30 min in the presence or absence of 1 μM Ko143 (***P < 0.001) (D). Initial velocity (5 min) of [3H] gefitinib uptake was measured after 4 hours of 0.1 μM gefitinib treatment (E). Each bar represents the mean (± SD) of four independent determinations. (** P < 0.01).