STAT3 modulates C/EBPα and C/EBPβ recruitment to the c-myc promoter in response to G-CSF. (A) A schematic diagram shows the location of putative STAT3, C/EBPα, and C/EBPβ binding sites in the murine c-myc promoter. (B) Sequence alignment of the murine, rat, and human c-myc promoter regions encompassing the putative STAT3, C/EBPα, and C/EBPβ binding sites. (C) Bone marrow cells from wild-type (WT) or STAT3-deficient (KO) mice were treated with G-CSF (25 ng/mL) for 1 hour or 6 hours, as indicated. ChIPs were performed with antibodies to STAT3, C/EBPβ, C/EBPα, or an irrelevant IgG (Ig). PCR reactions were performed with primers specific for the murine c-myc promoter region encompassing the putative STAT3, C/EBPα, and C/EBPβ binding sites, using immunoprecipitation or input samples, as indicated. Data are representative of 2 independent experiments. (D) Wild-type (WT) or STAT3-deficient (KO) mice were treated with G-CSF or BSA buffer in vivo. C/EBPα expression was determined in bone marrow Gr-1⁺ granulocytes at 6 hours after treatment by immunoblotting (n = 3 for WT and KO for each condition). Data are representative of 3 independent experiments. (E) 32D.G-CSFR cells were electroporated with pGL3-myc, pTK-Renilla, pMX-STAT3, and pMX-STAT3 DN as indicated. pRV-C/EBPβ or pRV-C/EBPα were included individually in some samples or in a 9:1, 1:1, or 1:9 ratio, as illustrated. Cells were treated with or without G-CSF for 2 hours and assayed for luciferase activity. Relative luciferase unit induction in G-CSF–treated vs unstimulated (NT) cells was averaged from 3 independent experiments. Error bars represent SEM.