AR knockdown in WT myeloid progenitors by RNA interference (RNAi) suppresses neutrophil differentiation, and restoring AR in ARKO cells induces neutrophil differentiation. (A) Bone marrow cells were plated in methylcellulose-containing media supplemented with the indicated cytokine and hematopoietic colonies containing >30 cells were scored after 7 d. We analyzed bone marrow cells from five 9-wk-old animals of each genotype. (B) Effect of adding various concentrations of DHT on colony formation of mouse bone marrow cells in methylcellulose-containing media supplemented with G-CSF. (C) The populations of GMPs and CMPs are comparable in bone marrow between WT and ARKO mice. GMPs were further isolated from WT and ARKO bone marrow using FACS sorting. (D) Representative photomicrographs of isolated neutrophils and precursors from bone marrow stained with Wright-Giemsa. A decrease of nuclei-segmented neutrophils (arrow heads) in ARKO bone marrow is observed when compared with the control. Bar, 10 µm. (E) GMPs were infected with retroviruses carrying pBabe vector, pBabe-AR, pSuperior vector, pSuperior-siAR, and pSuperior-scramble. Transduced GMPs were cultured in the presence of G-CSF (10 ng/ml) with or without 10 nM DHT for 7 d. Green fluorescent colony cells were then collected and counted. 200-count manual leukocyte differentials were examined after Wright-Giemsa staining. Numbers of neutrophils and precursor cells were calculated by multiplying neutrophil differential ratios by the total green fluorescent cell count. Triplicate experiments were performed and the error bars represent the SD.