In vitro and in vivo effects of G-CSF on CXCR4 and Gfi-1 expression in primary granulocytic lineage cells. (A) CXCR4 and Gfi-1 mRNA levels measured by real-time RT-PCR in Gr1⁺ bone marrow cells cultured in vitro with G-CSF for 1 hour to 6 hours. The results reflect the relative change in mRNA levels after culture compared with before culture; representative of 3 experiments. (B) CXCR4, Gfi-1, and actin levels detected by immunoblotting in Gr1⁺ bone marrow cells cultured in vitro with or without G-CSF for 1.5 to 6 hours; representative of 3 experiments. (C) CXCR4, Gfi-1, and actin content in cell lysates of Gr1⁺ bone marrow-derived cells after 18 hours of culture with G-CSF detected by Western blotting. The results reflect 3 independent experiments. Relative ratios of CXCR4/actin and Gfi-1/actin are shown in the bottom bar graph. (D) RNA was extracted from bone marrow Gr1⁺ cells from control mice, and mice were injected once with G-SCF 5 and 18 hours earlier. CXCR4 and Gfi-1 mRNA levels detected by real-time RT-PCR. The results reflect the relative change in mRNA levels in Gr1⁺ cells from mice treated with G-CSF compared with control. (E) Cells lysates were prepared from Gr1⁺ cells of control mice, and mice were injected once with G-CSF 5 and 18 hours earlier. CXCR4, Gfi-1, and actin levels were detected by immunoblotting. (F) After the mice were treated with G-CSF or diluent daily for 5 days, Gr1⁺ cells were purified from the bone marrow, and their content of CXCR4, Gfi-1, and actin were evaluated by Western blotting. The results are from 3 independent experiments. Relative ratios of CXCR4/actin and Gfi-1/actin are shown in the bottom bar graph.