Nutlin-3 inhibits TNF-α–induced NF-κB recruitment to the OPG promoter. (A) HUVECs were transfected with pOPG-Luc or empty pMetLuc-Reporter vector. After recovery, cells were stimulated as indicated, and after 18 hours luciferase activity was assessed. Results, expressed as RLU, represent mean plus or minus SD of 3 independent experiments (*P < .05). (B) HUVECs were either left untreated or stimulated with TNF-α in the absence and presence of Nutlin-3. NF-κB–p65 DNA binding activity was determined at the indicated time points as absorbance values per microgram of cell lysate protein. Results are mean plus or minus SD of 3 independent experiments performed in duplicates (*P < .05, compared with TNF-α). (C) Cells were stimulated with TNF-α in the absence and presence of Nutlin-3. ChIP analysis was performed using anti-p65 or control IgG antibodies for immunoprecipitation followed by OPG promoter-specific PCR with primers flanking the consensus κB sites. The PCR products were subjected to electrophoresis on 2% agarose gel. These results are representative of 3 independent experiments.