G-CSF–triggered phosphorylation of STAT5 is elevated in myeloid cells of CN patients; caSTAT5a inhibits mRNA expression of LEF-1 and its target gene C/EBPα. The levels of phospho-STAT5 in myeloid cells were assessed by FACS analysis of CD34⁺ cells from healthy individuals (n = 4), CN patients harboring ELA2 (n = 3) or HAX1 (n = 3) mutations, and CN patients who developed AML (n = 3) after incubating with or without recombinant human G-CSF (10 ng/mL) for 10 minutes. Cells were immunostained with an anti-phospho-STAT5 (Y694) antibody and isotype control antibody. (A) Representative histograms showing phSTAT5 (blue) and isotype (red) staining are depicted. (B) Bars show mean fluorescence intensity (MFI) of phospho-STAT5 (Y694) staining in CD34⁺ cells treated with G-CSF as described previously. Data represent means ± standard deviation (SD) and are derived from 2 independent experiments, each in triplicate (*P < .05, **P < .01). (C) SOCS3 mRNA expression in CD33⁺ cells from G-CSF–treated healthy individuals (n = 3) and G-CSF–treated CN patients (n = 6) was measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). SOCS3 mRNA levels were normalized to those of β-actin and are presented as arbitrary units (AUs). Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05). (D) G-CSFR mRNA and G-CSFR protein surface expression in CD33⁺ cells of G-CSF–treated healthy individuals (n = 3) and G-CSF–treated CN patients (n = 6). mRNA expression was measured by qRT-PCR. G-CSFR mRNA levels were normalized to those of β-actin and are expressed as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05). (E) CD34⁺ hematopoietic progenitor cells were transduced with a retroviral expression construct for caSTAT5a, WT STAT5a, STAT5a Y-F MUT, or ctrl-rv. After 60 hours of culture in X-VIVO 10 medium (Cambrex) supplemented with 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL thrombopoietin, 50 ng/mL stem cell factor, 50 ng/mL Flt3 ligand, and 10 ng/mL G-CSF, green fluorescent protein–positive cells were sorted, and LEF-1 and C/EBPα mRNA levels were measured by qRT-PCR. LEF-1 and C/EBPα mRNA levels were normalized to those of glyceraldehyde-3-phosphate dehydrogenase and are expressed as AUs. G-CSFR surface expression was measured by FACS. Data represent means ± SD and are derived from 2 independent experiments, each in triplicate (*P < .05, **P < .01). (F) CD34⁺ cells from 1 CN patient were incubated with 5 μM pimozide, a specific STAT5 inhibitor, for 48 hours, and LEF-1 mRNA expression was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are expressed as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in duplicate (*P < .05). DMSO, dimethylsulfoxide.