IFN-γ reduces TPO-mediated STAT5 phosphorylation. (A) Histogram showing phosphorylated STAT5 staining of purified HSCs (Lin⁻c-Kit⁺CD48⁻CD150⁺) cultured for 24 hours with SCF (shaded histogram), SCF and TPO (bold histogram), or SCF, TPO, and IFN-γ (thin histogram). Percentage of cells stained positive for pSTAT5 (B) and geometric mean fluorescence intensity of pSTAT5 (C). Data represent triplicate cultures and are representative of 2 independent experiments. (D) Purified HSCs were cultured as in Figure 1 with or without IFN-γ and/or TPO and HSC numbers were measured. Data are representative of 2 experiments with 4 wells per condition. (E) Histogram showing pSTAT1 staining of purified LKS cells stimulated for 45 minutes with SCF (shaded histogram) or SCF and IFN-γ (bold histogram). Histograms are representative of 4 independent experiments. (F) Messenger RNA expression levels of IRF-1 and SOCS1 in HSCs cultured for 24 hours with SCF or SCF and TPO with or without IFN-γ. Expression levels are relative to the expression in SCF cultured HSCs. Quantitative PCRs were performed in duplicate and data are pooled from 2 independent experiments with 1 to 3 cultures per condition. (G) Plots showing pSTAT5 staining of lineage-depleted BM cells after retroviral transduction with a vector encoding SOCS1 or with an empty vector as a control. Cells were cultured for 48 hours followed by TPO stimulation with or without IFN-γ and intracellular staining for pSTAT5. pSTAT5 staining of transduced (green fluorescent protein positive) cells is shown. Plots are representative of 3 independent experiments. Mean values ± SD are shown. *P < .05; **P < .01; ***P < .001. GeoMFI, geometric mean fluorescence intensity.