2006-PO down-regulates the expression of EPOR and EKLF. (A) Quantitative analysis of EPOR using real time RT-PCR. Purified CD34⁺ cells were cultured for 3 days in erythroid medium and the cells enriched as CD71⁺ cells. The CD71⁺ cells were then cultured in erythroid medium, with or without 2006-PO. At the indicated days, the cells were harvested and mRNA extracted from the cells and subjected to real-time RT-PCR. (B-C) Fluorocytometric analysis of EPOR expression. At the indicated days, the cells were harvested and EPOR expression on GPA⁺ cells analyzed using fluorocytometry. Data are representative of 3 independent experiments (B) and are mean ± SD (C left panel). (C right panel) Parallel experiments in which 2006-PO was replaced with ODN 5′-GTTTTGT-3′. (D) Specific binding of ¹²⁵I-EPO. Purified CD34⁺ cells were cultured for 3 days in erythroid medium and the cells were then cultured in erythroid medium, with or without 2006-PO. At the indicated days, the cells were harvested and subjected to ¹²⁵I-EPO binding. The cells were collected in IMDM at 0°C containing 0.1% BSA, and 4 × 10⁵ cells in 50 μL were incubated in the presence of 2 U/mL ¹²⁵I-EPO. Data are mean ± SD of triplicate measurement; representative data of 2 experiments are shown. (E) Quantitative analysis of EKLF transcripts by real-time RT-PCR. CD71⁺ cells were incubated in erythroid medium with or without 2006-PO, the cells harvested, and mRNA extracted and quantified by real-time RT-PCR. (F) Western blot analysis of EKLF. The cells were harvested from the parallel experiments shown in panel E, and the lysates separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nylon membranes. Western blot analyses were performed using an anti-EKLF antibody. (G) Relative expression of EKLF. Data are the mean ± SD of 3 independent experiments. *P < .05. ***P < .001. NS indicates no significance.