Analysis of BM neutrophils from mice injected with G-CSF.

(A) Phenotypic analysis of BM cells from WT and Ik^L/Lmice injected over 5 days with G-CSF or PBS (control). Cells were analyzed 2 hours after the last injection for their expression of Mac-1 and Gr-1 (top). Line graphs compare the intensity of the Gr-1 staining between WT and Ik^L/L cells for each condition. (B) Semiquantitative RT-PCR of transcripts encoding transcription factors (C/EBPε and STAT3), secondary granule markers (gelatinase, lactoferrin, and lysozyme), and Ly-6G in WT and Ik^L/L BM cells. Each panel represents ethidium bromide-stained products from PCR reactions corresponding to increasing numbers of cycles. The numbers of PCR cycles were 24, 27, 30 (C/EBPε, gelatinase, lysozyme, Ly-6G, STAT3); 27, 30, 33 (lactoferrin); and 18, 21, 24 (β-actin). (C) G-CSF induction of STAT3 binding. Electrophoretic mobility shift assay performed with 3 μg of nuclear extracts from BM cells derived from mice injected or not with G-CSF. Binding was performed using probes corresponding to WT or mutated (right lanes) STAT3- or PU.1-binding sites. The asterisk indicates a nonspecific band binding the mutated PU.1 site.