IMiDs down-regulate PU.1 in myeloid progenitors in vitro and in patients. Purified CD34⁺ cells were cultured in the presence of DMSO (control), (A) pomalidomide, or (B) lenalidomide for 6 and 8 days, and PU.1 protein expression was determined. β-Actin served as loading control. (C) CD34⁺ cells were cultured in the presence of DMSO (control), pomalidomide, or lenalidomide for 3 days. Total RNA was extracted and then subjected to quantitative RT-PCR. Data were analyzed according to the ΔC_T method. Results are shown as mean CG mRNA fold increase by pomalidomide or lenalidomide compared with baseline (control). The level of mRNA was normalized to GAPDH expression. (D) Double labeling immunohistochemistry on paraffin-embedded bone marrow biopsy sections for PU.1 expression (black nuclear staining) and myeloperoxidase (red cytoplasmic staining) before and during treatment with either lenalidomide or dexamethasone (control). Slides were evaluated using an Olympus BX45 microscope equipped with a 100×/1.35 numeric aperture (Olympus; original magnification ×1000).