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Effect of ABCG2 silencing on [3H]gefitinib accumulation, efflux, and uptake.

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posted on 04.11.2015, 06:20 by Maricla Galetti, Pier Giorgio Petronini, Claudia Fumarola, Daniele Cretella, Silvia La Monica, Mara Bonelli, Andrea Cavazzoni, Francesca Saccani, Cristina Caffarra, Roberta Andreoli, Antonio Mutti, Marcello Tiseo, Andrea Ardizzoni, Roberta R. Alfieri

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).