G-CSF induced STAT3 signaling and Gfi-1 expression. (A) Western blot analysis of STAT1, STAT3, and STAT5 phosphorylation in cell lysates of 32Dcl3 cells cultured for 1 to 144 hours with IL-3 alone or with IL-3 plus G-CSF. Blots were first stained for phosphorylated Stat1 and then stripped and reprobed for P-STAT3, PSTAT5, and total STAT3. (B) Semiquantitative RT-PCR analysis of CXCR4, Gfi-1, and GAPDH mRNAs in 32Dcl3 cells before subculture (time 0) or after incubation with IL-3 alone or with IL-3 plus G-CSF for 24 and 96 hours. Representative experiment (n = 5). (C) Real-time RT-PCR analysis of CXCR4 and Gfi-1 mRNA in 32Dcl3 cells after 24 and 96 hours of incubation with IL-3 alone or with IL-3 pus G-CSF. The results reflect the mean fold (± SD) mRNA change in cells cultured with IL-3 plus G-CSF relative to cells cultured in IL-3 alone. (D) Kinetics of G-CSF–induced Gfi-1 protein expression evaluated by Western blotting. 32Dcl3 cells were cultured for 24 to 144 hours with IL-3 alone or with IL-3 plus G-CSF. The blots were stripped and restained for actin. Relative ratios of Gfi-1 to actin are shown in the lower bar graph.