STAT3 controls c-myc expression during emergency granulopoiesis. (A) Wild-type (WT) or STAT3-deficient (KO) mice were treated with G-CSF or BSA as indicated in Figure 1. c-myc mRNA expression was determined in bone marrow LSK and GMP subsets isolated 24 hours after treatment or in immature Gr-1lo granulocytes at 4 hours after treatment, by quantitative PCR (n = 3 for WT and KO for each condition). (B) WT or STAT3-deficient mice were infected with L monocytogenes or left untreated. c-myc expression was analyzed in LSKs and GMPs purified 12 hours after infection, using quantitative PCR (n = 3 for WT and KO for each condition). (A-B) Average values from 3 independent experiments are shown. *P < .05, **P < .01, ***P < .001 compared with control. (C) 32D.G-CSFR cells were treated 0 to 6 hours with G-CSF; c-myc RNA was measured by quantitative PCR. Average values from 3 independent experiments are shown. ***P < .001 compared with control. (D) Nuclear extracts were generated from untreated (−) or G-CSF–treated (+) 32D.G-CSFR cells at the indicated times and used in EMSAs with a radiolabeled oligonucleotide corresponding to a putative STAT3 consensus site in the c-myc promoter (WT), a mutated radiolabeled c-myc promoter oligonucleotide (mutant), or a radiolabeled oligonucleotide corresponding to the STAT3 binding site in the murine Socs3 promoter (S). EMSAs were performed in the presence or absence of STAT3 antibodies or competitor WT oligonucleotides, as indicated. The migration positions of STAT3 dimers (*) and supershifted STAT3 complexes (**) are shown. Data are representative of 3 independent experiments. (E) 32D.G-CSFR cells were treated as indicated in Figure 4E. ChIPs were performed with STAT3 antibodies (STAT3) or an irrelevant IgG (Ig), as indicated, followed by PCR with primers specific for the murine c-myc promoter. Control PCR reactions were performed on total lysate (input). Data are representative of 3 independent experiments.
