Restoration of defective LEF-1 expression and activity by treatment of CN CD34⁺ progenitors with bortezomib. In vitro G-CSF–triggered granulocytic differentiation of CD34⁺ cells from CN patients and healthy individuals was performed in the presence or absence of bortezomib (10 nM), as described in Materials and Methods. (A) mRNA expression of LEF-1 and the LEF-1 target genes C/EBPα and cyclin D1 in studied groups, as assessed by RT-PCR. LEF-1, C/EBPα, and cyclin D1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (*P < .05, **P < .01). (B) Intracellular expression levels of LEF-1 protein detected using FACS analysis, as described in “Materials and methods”. Representative histograms as well as percentages of positive cells are depicted. (C) CD34⁺ cells from healthy individuals were transduced with caSTAT5a or ctrl rv constructs and incubated with or without bortezomib (10 nM). After 26 hours of treatment, cells were sorted and LEF-1 mRNA expression was measured by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 2 independent experiments, each in duplicate (*P < .05).