IMiD compounds downregulate the IKZF1 protein in human CD34⁺ cells. (A) The protein expression of CRBN, IKZF1, and IKZF3 was analyzed in human myeloma cell lines (RPMI-8226, H929, and MM.1S) and human CD34⁺ cells (Patients 1 and 2) by western blotting. (B) CD34⁺ cells were cultured with either LEN or POM at the indicated concentrations for 24 hours (upper) or for a short period (1-24 hours at 1 μM; bottom) or (C) a long period (1-9 days at 1 μM). Drugs were added every 2 days. The cell lysates were analyzed for IKZF1 and CRBN expression by western blotting. β-Actin was used as a loading control. (D) CD34⁺ cells were treated with DMSO (0.01%), LEN, or POM (1 μM) for 6 hours. The cells were then fixed and stained for IKZF1 (red color), and nuclear counterstaining was performed with DAPI (blue color). The localization of IKZF1 was observed using a Leica microscope (40×). (E) CD34⁺ cells were treated with DMSO (0.01%), LEN, or POM (1 μM) for the indicated times. IKZF1 mRNA levels were compared using qRT-PCR. (F) For thalidomide analog bead pull-down assays, we used a thalidomide analog coupled with FG beads (high-performance magnetic nanoparticles) to pull down the bound protein, followed by immunoblot detection of CRBN. Input, 5% of total protein from CD34⁺ cells before affinity bead binding; DMSO, LEN, or POM, pull-down complex from CD34⁺ cell extracts preincubated with DMSO (0.01%), lenalidomide, or pomalidomide (10 μM) for 15 minutes at room temperature.