Characterization of BCRP downregulation by imatinib. (A,C) Cells were exposed to imatinib under the concentrations indicated at 37°C for 14 hours (A) or the times indicated (C). Total cell lysates were then extracted and subjected to Western blotting. These Western blots represent 3 individual experiments done on different days. (B) Cell growth relative to control (without imatinib) was determined by counting cell number with a Coulter Counter (n > 3). (D) Effects of pre-exposure to imatinib on BCRP function. K562/BCRP-MX10 cells were cultured with 0 or 5 μM imatinib for 14 hours, and then the cells were washed free of drug and cultured for an additional 80 minutes in fresh medium containing 10 μM mitoxantrone. At this point, intracellular mitoxantrone content was determined using flow cytometry. Each bar represents the mean value ± SEM for 4 individual experiments. The difference between imatinib-treated and control cells was evaluated by Student t test. (E) Relative BCRP mRNA expression was quantified by 4 individual qRT-PCR experiments. Each vertical bar represents the mean value ± SEM.