Figure 7.
Mitoxantrone accumulation in mutant breast cancer resistance protein (BCRP) transfectants. (a) Cells (5 × 105) were incubated in the presence or absence of 1 μM mitoxantrone for 40 min at 37°C and then washed and resuspended in ice‐cold PBS. Fluorescence was determined by flow cytometry. Bars indicate median channels of fluorescence in the transfectants. (1) PA317 cells; (2) PA/WT‐mix cells; (3) PA/C592S‐cl.6 cells; (4) PA/C603S‐cl.3 cells; (5) PA/C592S·C608S‐cl.7 cells; (6) PA/C592S·C603S·C608S‐cl.7 cells. (b) Cells were seeded onto 60 mm dishes (2 × 105 cells/dish) and cultured overnight. Cells were transfected with non‐silencing control or BCRP siRNA using Lipofectamine 2000. Western blot analysis was performed under non‐reducing conditions (−SH). GAPDH expression was analyzed as an internal control of protein loading. (c) The mitoxantrone accumulation assay was performed in siRNA‐transfected cells as described in (a). (1) PA317 cells transfected with control siRNA. (2) PA317 cells transfected with BCRP siRNA. (3) PA/WT‐mix cells transfected with control siRNA. (4) PA/WT‐mix cells transfected with BCRP siRNA. (5) PA/C592S·C603S·C608S‐cl.7 cells transfected with control siRNA. (6) PA/C592S·C603S·C608S‐cl.7 cells transfected with BCRP siRNA.