DBY colocalized with HLA class II molecules in male leukemic cells. (A) Flow cytometry of HLA-DR expression of leukemic cell lines (HEL, KasumiA-541, KHM-2B, KO52, Raji, and THP-1 cells). Cells were stained with isotype control (shaded histogram) and anti-HLA-DR (red line) antibodies. (B) Quantitative real-time polymerase chain reaction analysis of the expression of DBY messenger RNA (mRNA) levels in the leukemic cell lines. Bar plots indicate DBY mRNA expression relative to control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. DBY mRNA levels are presented as the mean ± SD of 3 independent experiments. (C) PLAs to visualize colocalization between DBY and HLA-DR in leukemic cell lines. Colocalization of DBY and HLA-DR induced PLA signals (red). Leukemic cell lines of KO52 (left) and THP-1 (right). (D) Cryopreserved isolated mononuclear cells from bone marrow (BM) in patients with leukemia at diagnosis were used for PLAs. Patients 1 and 2 were diagnosed with HLA-DR–positive acute myeloid leukemia (AML). Wright-Giemsa stain (upper 2 microphotographs) and PLAs (lower 2 fluorescence microphotographs). Nuclei were stained with DAPI. Scale bars: 20 μm (C-D). Original magnification, ×400 (C-D). All data are representative of 3 independent experiments. DAPI, 4',6-diamidino-2-phenylindole.