STAT3 directly regulates C/EBPβ expression during emergency granulopoiesis. (A-B) C/EBPβ expression was measured in Gr-1⁺ granulocytes isolated from wild-type (WT) and STAT3-deficient (KO) mice 6 hours after treatment with G-CSF (+) or BSA buffer (−) in vivo (A) or 24 hours after infection with L monocytogenes (+) or in untreated controls (−) (B). The dominant LAP (liver-enriched transcriptional activator protein) isoform of C/EBPβ was detected by immunoblotting. Tubulin was used as a loading control. Data are representative of 3 independent experiments. (C) Cebpb mRNA expression was measured in immature Gr-1^lo granulocytes from WT or STAT3-deficient mice, after treatment with G-CSF ex vivo for 0 to 4 hours, using quantitative PCR (n = 3 for WT and KO for each condition). Average values from 3 independent experiments are shown. **P < .01 compared with control. (D) Schematic diagram of the murine Cebpb promoter, showing the location of 2 IL-6 RE II sites. (E) 32D.G-CSFR cells were stimulated with G-CSF (25 ng/mL) for 1 hour or left untreated, as indicated. Nuclear extracts were used in EMSAs with a radiolabeled oligonucleotide corresponding to the IL-6 RE II consensus sequence at position −1180 in the Cebpb promoter (C/EBPβ probe) or a mutant radiolabeled C/EBPβ oligonucleotide (mutant C/EBPβ) in the presence or absence of unlabeled competitor C/EBPβ probe (C), an unlabeled competitor oligonucleotide corresponding to the STAT3 consensus site in the murine Socs3 promoter (S), or STAT3 antibodies, as indicated. The migration positions of STAT3 dimers (*) and supershifted STAT3 complexes (**) are shown. Data are representative of 3 independent experiments. (F) 32D.G-CSFR cells were electroporated with pGL3-C/EBPβ, pTK-Renilla and pMX-STAT3 or pMX-STAT3 DN (DNA-binding mutant). After 24 hours, cells were treated with or without G-CSF for 2 hours and assayed for luciferase activity. The ratio of firefly:renilla relative light units (RLU) from G-CSF–treated and unstimulated cells (NT) was averaged from 3 independent experiments. Error bars represent SEM. (G) 32D.G-CSFR cells were treated as indicated in panel E. ChIPs were performed with STAT3 antibodies (STAT3 Ab) or an irrelevant IgG (Ig). PCR reactions were performed with primers specific for the murine Cebpb promoter on total cell lysates (input) or immunoprecipitated samples, as indicated. Data are representative of 3 independent experiments. (H) Wild-type Gr-1^lo cells were infected with lentiviral shRNA vectors (control vector, gray shading; shRNA 1, dotted line; shRNA 2, black line), stained with CFSE, analyzed immediately (NT) by flow cytometry or cultured in G-CSF for 4 days (G-CSF), and evaluated by flow cytometry. Expression of C/EBPβ, C/EBPα, and tubulin was determined by immunoblotting. Data are representative of 3 independent experiments.