Figure 4.

The IMiD-induced myeloid lineage shift of CD34⁺ cells is mediated by IKZF1. CD34⁺ cells were transduced using a lentivirus carrying the control shRNA (shCNTL), IKZF1-shRNA #1 (shIKZF1-1), or IKZF1-shRNA #2 (shIKZF1-2) sequence. At 3 days after transduction, the cells were sorted, and (A) the lysates were analyzed by western blotting to compare the levels of IKZF1. (B) The sorted cells were treated with POM (1 μM) or DMSO (0.01%) as a control for 6 hours, then fixed with 4% formaldehyde and stained for IKZF1 (red color) and GFP (green color) and with DAPI for nuclear counterstaining (blue color). The localization of IKZF1 was observed using a Leica microscope (200×). (C) shCNTL, shIKZF1-1 and shIKZF1-3 CD34⁺ cells were treated with DMSO (0.01%) or POM (1 μM) and subjected to colony-formation assays. After 14 days, the BFU-E and CFU-G colony numbers were counted. (D-E) CD34⁺ cells were transduced with empty vector (EV), PCDH-IKZF1-WT (IKZF1^WT) or PCDH-IKZF1-Q146H (IKZF1^Q146H) and sorted based on GFP expression after 3 days. (D) Sorted cells were treated with POM (1 μM) or DMSO (0.01%) for 6 hours. Cell lysates were analyzed by western blotting to compare levels of IKZF1. (E) Sorted cells were analyzed for GFP⁺, CD33⁺, and CD45⁺ by flow cytometry for the evaluation of myeloid differentiation. (F) Sorted cells were treated with DMSO (0.01%) or POM (1 μM) and subjected to colony-formation assays. After 14 days, the colonies were counted using a Leica microscope (25×). *P ≤ .05; **P ≤ .01.