Stat3 transcriptional activity is reduced in ARKO neutrophils. (A) PIAS3, Stat3, and pY-Stat3 proteins are comparable in WT and ARKO bone marrow neutrophils. Cells were isolated from mice and then incubated with G-CSF (40 ng/ml) at 37°C for the indicated period. (B) AR protein expression is lower in bone marrow neutrophils of castrated mice than those of WT mice, while Stat3 and pY-Stat3 levels are comparable. Bone marrow Gr-1⁺ cells were isolated from mice and then incubated with G-CSF (40 ng/ml) at 37°C for 30 min. (C) AR interacts with the GST-PIAS3 in the GST pull-down assay. (D) Binding of Stat3 by PIAS3 is more in ARKO bone marrow neutrophils. The result of band intensities of Stat3, pY-Stat3, and AR in the coimmunoprecipitation experiment was normalized with band intensities of PIAS3 and shown in the right panel. (E) AR can relieve the PIAS3 inhibition of activated Stat3 action. The results are presented as fold induction of luciferase activity from triplicate experiments using CV-1 cells and the error bars represent the SD. ***, P < 0.001. (F) CA-Stat3 induces but DN-Stat3 inhibits neutrophil maturation. (G) PIAS3 inhibits neutrophil maturation, while increased AR rescues the inhibition. (H) PIAS3-siRNA increases neutrophil maturation. Results are presented as cell numbers of neutrophils and precursors from triplicate experiments and the error bars represent the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.