IMiDs induce cereblon-dependent degradation of aromatase. (A-B) Protein expression levels of 3β-HSD (A) and aromatase (AROM) (B) in megakaryocytes generated from CD34⁺ HSPCs. (C) Relative mRNA expression levels of CYP19A1 in megakaryocytes generated from CD34⁺ HSPCs. The cells were treated with dimethyl sulfoxide, 10 µM of lenalidomide (Len), or pomalidomide (Pom) for 24 hours. (C) Data are from 3 independent experiments and expressed as means ± standard errors of the mean. P values were determined by 2-tailed Student t test. (D) Time course of 10-µM Len treatment in megakaryocytes for AROM protein levels. (E-F) Immunoblot analysis for AROM protein levels in megakaryocytes treated for 24 hours with Len (E) or Pom (F) at indicated concentrations. (G) Immunoblot analysis for AROM protein levels in K562 cells treated for 24 hours with or without 10 µM of IMiDs alone or in the presence of 10 µM of MG132 or 1 µM of MLN4924. (H) Lysate mixture of K562 cells transfected with HA-tagged AROM and those transfected with FLAG-tagged cereblon (CRBN) was incubated with or without 10 µM of Len for 12 hours and subjected to immunoprecipitation with antibody against HA. (I) FLAG-tagged CRBN was immunoprecipitated from the lysates of 293T cells transfected with indicated CRBN variants and used to capture AROM from 293T cells transfected to overexpress aromatase. (J) Purified recombinant His-tagged CRBN-loaded beads were incubated with glutathione S-transferase (GST)–tagged AROM in the presence of Len at indicated concentrations. Results are representative of 2 (A, G, H, I, J) or 3 (B, D, E, F) independent experiments. Cont, control; IB, immunoblotting; IP, immunoprecipitation.