miR-223 functions as a tumor suppressor in acute myeloid leukemia. (A) Quantitative real-time RT-PCR for miR-223 was carried out using bone marrow cells derived from AML patients. Values were normalized with U6. Cord blood (CB) and peripheral blood (PB) and AML with normal karyotype (nk), with complex karyotype (ck), with FLT3 mutation (FLT3 mut), and with CEBPA mutation (CEBPA mut). Data are represented as mean from 3 experiments. (B) U937 cells were transfected with lentiviral control or lenti-223 vectors. Two days later, total RNA was isolated and analyzed for miR-223 by quantitative real-time RT-PCR. Data are represented as mean ± SD from 3 independent experiments. **P < .01. (C) Growth curve of U937 cells transfected with lentiviral control or lenti-223 vectors. Data are represented as mean ± SD from 3 independent experiments. **P < .01. (D) Analysis of cell-cycle distribution during miR-223 lentiviral overexpression by flow cytometry. Representative FACS data from U937 cells transfected for 2 days with control and miR-223 lentiviral vector. (E) Flow cytometry of BrdU/PI-stained U937 cells transfected with lentiviral control or lenti-223 vectors from a representative experiment. Two days after lentiviral transfection, cells were labeled with BrdU for 45 minutes. Cells were then fixed, stained with FITC-labeled anti-BrdU antibody and PI, and analyzed by flow cytometry to determine the cell-cycle distribution. Bivariate analysis of the incorporation of BrdU (BrdU FITC, vertical axis) and the DNA content (FL3, PI staining, horizontal axis) was performed.