Nutlin-3 down-modulates OPG release in endothelial cells. HUVECs were left untreated or treated with Nutlin-3 (10 μM) or Aphidicolin (1 μg/mL) for 24 hours. (A) Comparison between the effects of Nultin-3 and Aphidicolin on the p53 pathway and cell cycle. Levels of p53 and p21 proteins were assessed by Western blot analysis in cell lysates. Tubulin staining is shown as loading control. In parallel, cell cycle was analyzed by BrdU labeling and PI staining. In each panel, the rectangle represents the cells in S phase of the cell cycle, which have incorporated BrdU. Representative examples of 3 to 6 independent experiments of Western blot results and cell-cycle profiles analyzed by flow cytometry. (B) OPG levels in culture supernatants were assessed by ELISA. Data are mean plus or minus SD of results from 4 independent experiments, each performed in duplicate (*P < .05, compared with untreated).