HU activates NF-κB signaling and enhances NF-κB binding to the SAR1 promoter region. (A) EMSA for the Elk-1/NF-κB was performed using K562 nuclear extracts (10 μg) and oligonucleotide probes containing either a wild-type or mutant Elk-1/NF-κB–binding site. Competition analysis was performed in the presence of 10-, 100-, or 500-fold excess of unlabeled oligonucleotides (right panel). Antibody-supershift assays were performed using antibodies against NF-κB p50, c-Rel, and Elk-1. Two Elk-1/NF-κB–specific DNA-protein complexes are indicated as A and B. The DNA-protein complex supershifted by anti-NF-κB p50 antibody is indicated as ss. (B) EMSA analysis of the effects of HU on NF-κB binding to its recognition site in the SAR1 promoter. EMSA was performed using nuclear extracts (10 μg) isolated from K562 cells treated with 100μM HU for the indicated period and oligonucleotide probes containing the Elk-1/NF-κB–binding site. A and B represent Elk-1/NF-κB–specific DNA-protein complexes as described in A. (C) At day 6 of differentiation, CD34⁺ cells were treated with or without 100 µM HU. Cells were then harvested and subjected to ChIP assay using antibody against NF-κB or Elk-1 to immunoprecipitate chromatin-protein complexes. A parallel ChIP assay was performed using rabbit IgG for the immunoprecipitation step as a ChIP assay negative control. DNA was amplified and quantitated by PCR with specific primers flanking the SAR1 gene promoter from −137 to −12. (D) CD34⁺ cells were treated in the presence or absence of 100 μM HU from day 4 to day 7 of differentiation, and preincubated in medium with 0, 5, 7.5, or 10 μg/mL BAY11-7082 for 30 minutes at day 6, transfected with a construct containing the −977 to +49 region of the SAR1 gene, then assayed for luciferase activity 24 hours after transfection. *P < .05 vs mock-transfected cells without treatment. Mock-, SAR1 reporter construct–, and vector control–transfected cells are indicated as M, SAR1, and V, respectively. The level of promoter activity was evaluated by measurement of the firefly luciferase activity relative to the internal control Renilla luciferase activity using the Dual Luciferase Assay system. Error bars represent SD of the mean of 3 independent experiments. (E) At day 5 of differentiation, CD34⁺ cells were treated with or without 4 μg/mL BAY11-7082 in the presence or absence of 100 µM HU for 3 days, then harvested and measured for SAR1 (left panel) and γ-globin (right panel) expression by real-time PCR analysis. Fold increase was calculated relative to expression in cells without BAY11-7082 and HU treatment after normalization with β-actin gene expression. *P < .01 vs CD34⁺ cells without BAY11-7082 and HU treatment. **P < .01 vs CD34⁺ cells with HU treatment only. Error bars represent SD of the mean of 3 independent experiments. m, mutant; w, wild-type.