Effect of mutations in ABCG2 on drug efflux function. HEK293 cells with stable expression of ABCG2Myc-WT, ABCG2Myc-TM5*6*, ABCG2Myc-ECL3*, ABCG2Myc-TM5*L3*, ABCG2Myc-L3*TM6*, and vector-transfected control cells (Vec) were subjected to mitoxantrone (MTZ) accumulation analysis using flow cytometry as described in Materials and Methods. The relative accumulation of mitoxantrone was calculated after normalization to the expression level of ABCG2 and then to that of the vector-transfected control from three independent experiments. **p < 0.01.