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. 2018 Mar 1;2(5):492–504. doi: 10.1182/bloodadvances.2017010348

Figure 5.

Figure 5.

PU.1 is regulated by IKZF1 in CD34+ cells. (A) CD34+ cells were cultured with DMSO (0.01%) or POM (1 μM) for the indicated times, and compounds were added every 2 days. The cells were subsequently lysed and analyzed for PU.1 expression by western blotting. (B) CD34+ cells were treated with DMSO (0.01%), LEN, or POM (1 μM). IKZF1 and PU.1 mRNA levels were compared using qRT-PCR. (C) Chromatin immunoprecipitation analysis of DMSO (0.01%) or POM (1 μM)-treated CD34+ cells using an IKZF1 antibody or rabbit IgG as a negative control. The precipitated DNA fragments were subjected to qRT-PCR analysis with primers targeting the PU.1 promoter. (D) shCNTL, shIKZF1-1, and shIKZF1-3 CD34+ cell lysates were analyzed by western blotting to compare the levels of IKZF1, CRBN, and PU.1 (E) shCNTL and shCRBN- CD34+ cells were treated with DMSO (0.01%) or POM (1 μM) for 3 days. The cell lysates were then analyzed by western blotting to compare the levels of CRBN and PU.1. β-Actin was probed as a loading control. (F) shCNTL and shCRBN- CD34+ cells were treated with DMSO (0.01%) or POM (1 μM) for 24 hours and analyzed by chromatin immunoprecipitation assay using an IKZF1 antibody, or rabbit IgG as a negative control. The precipitated DNA fragments were subjected to qRT-PCR analysis with primers targeting the PU.1 promoter. **P ≤ .01.