ABCG2 expression and function in myeloma cell lines. (A) Flow cytometric analysis of MM cell lines for ABCG2 membrane expression was performed. 8226MR cells express more ABCG2 than wild-type 8226 cells, and H929 cells express very little, as shown by the shift in fluorescence. (B) Immunostaining of myeloma cell lines using an anti-ABCG2 FITC (Chemicon)–labeled antibody (ABCG2 is green and DAPI is blue). (C) Western blot of protein (25 μg/lane) extracted from myeloma cell lines for ABCG2. (D) Functional analysis of ABCG2 using topotecan as a substrate and the ABCG2-specific inhibitor tryprostatin A. Topotecan, a very good ABCG2 substrate and a naturally fluorescent molecule, is effluxed in high (8226MR) and moderate (8226) ABCG2 expressers, but is accumulated by H929 cells (which do not express ABCG2). Tryprostatin A (trypA) blocks the efflux of topotecan, demonstrating that topotecan efflux is ABCG2 dependent.