Exogenous MN1 inhibits the differentiation of AML cell lines toward monocytes and granulocytes. (A) Cytospin preprations of FACS-sorted GFP⁺ U937 and HL60 cell lines transduced with control GFP (mock) or MN1-expressing retroviruses (MN1), stained with anti-MN1 antibody (red). Blue represents nuclear staining with 4,6-diamidino-2-phenylindole. (B) MN1 expression in transduced HL60 and U937 cells was analyzed using real-time RT-PCR. Each sample was analyzed in triplicate, and the signals were normalized for endogenous HPRT expression. Histogram shows mean ± SEM of duplicates. MN1(−) indicates mock; MN1(+), MN1-transduced cells; and ND, not determined. (C) Sorted GFP⁺ HL60 cells were seeded at day 0 in duplicates in 6-well plates (10⁵ cells/mL, 4 mL/well) with indicated amounts of vitamin D₃, ATRA, or vehicle, and cells were counted at consecutive days (mean ± SEM of duplicates). ***P = .001. (D) Cells were treated with 1 μM ATRA or (E) with the indicated amount of vitamin D₃ or the same volume of vehicle in duplicates for 72 hours. Expression of CD11b and CD15 was analyzed by FACS. The result from 1 representative example is shown. (F) Low, medium (mid), or high GFP-expressing U937-MN1 cells were FACS-sorted (top left panel; GFP gates) and MN1 expression (normalized to HPRT) was compared with that of human CD34-BM cells and AML patients (1-6) using real-time RT-PCR (top right histogram). CD11b expression of cells treated for 48 hours with vehicle or the indicated amounts of vitamin D₃ or ATRA was determined using FACS in all GFP⁺ cells (bottom panels, blue, magenta and orange together; U937-mock and U937-MN1) or in GFP-low fractions (blue panels). All experiments were repeated a minimum of 3 times.