IFNγ reduces G-CSF–mediated STAT3 phosphorylation in a SOCS3-dependent manner. (A) Representative histograms showing intracellular staining for pSTAT3 in total BM cells stimulated for the indicated times with G-CSF with or without 45 minutes of preincubation with IFNγ. (B) Representative histograms showing overlays of intracellular staining for pSTAT3 in total BM cells and GMPs after 30 minutes of stimulation with G-CSF with or without preincubation with IFNγ. (C) Relative expression of SOCS3 in WT GMPs after 30 minutes in vitro stimulation with IFNγ measured by quantitative PCR analysis. Expression is relative to the levels in unstimulated GMPs. Data represent means ± SD from triplicate cultures. (D) Representative histograms showing overlays of intracellular staining for pSTAT3 in 32D cells expressing WT or a mutant human G-CSFR after 30 minutes of stimulation with G-CSF with or without preincubation with IFNγ. The SOCS3-recruiting tyrosine motif Y729 is only present in cells expressing WT G-CSFR or the mutant receptor denoted as Y729. All data are representative of at least 2 independent experiments. ***P < .001.