Insensitivity to G-CSF signaling in ARKO neutrophils. (A) Only few apoptotic neutrophils can be found in freshly isolated WT and ARKO bone marrow cells. (B) G-CSF protection against apoptosis in ARKO bone marrow neutrophils was less than in WT cells in vitro. Isolated mature bone marrow neutrophils were cultured in RPMI medium with 20% FCS without or with G-CSF (100 ng/ml) for 48 h. Neutrophils were then stained with Annexin V and 7-AAD, and analyzed by flow cytometry. (C) A lower increasing rate of blood neutrophils was found in ARKO mice in response to G-CSF injection. (D) G-CSF target genes are down-regulated in ARKO neutrophils. Data represent mean ± SD for three separate experiments. *, P < 0.05; **, P < 0.01. (E) SOCS3 protein expression is decreased in ARKO neutrophils. (F) mRNA expression of G-CSFR is comparable between WT and ARKO neutrophils. (G) G-CSF binding to WT and ARKO neutrophils is similar.