AR is essential for ERK1/2 activation in G-CSF induced neutrophil proliferation. (A) With G-CSF (40 ng/ml) treatment for the indicated times, ERK1/2 phosphorylation status was examined in bone marrow neutrophils of WT, castrated WT, and ARKO mice. (B) AR is required for G-CSF-induced ERK1/2 phosphorylation. By immunoblotting, the expression status of phosphorylated ERK1/2 and total ERK1/2 were compared among each group of G-CSF–induced granulocytes derived from WT or ARKO bone marrow cells infected with retroviruses carrying AR, AR-siRNA, or control vectors. v, vector alone; Sc, pSuperior-scrambled siRNA; Si-AR, pSuperior-AR siRNA. (C) U0126 almost abolishes G-CSF–induced proliferation of bone marrow cells. In the presence or absence of G-CSF (10 ng/ml), bone marrow cells (106/ml) were incubated with or without 1 µM U0126 for 3 d. Proliferative activity was then examined by 3H-thymidine incorporation. (D) Effects of AR deficiency on the human myeloblastic cell line, KG-1. Stably transfected cells were plated at 5 × 105 cells/ml in RPMI medium and the number of total viable cells was calculated each day. Results are the mean of three independent experiments and the error bars represent the SD. (E) Schematic representation of the role of AR in G-CSF signaling.
