Nutlin-3 down-modulates OPG release in a p53-dependent manner. HUVECs were transfected with control scrambled siRNA or p53 siRNA before treatment with Nultin-3 (10 μM) or TNF-α in the absence and presence of Nultin-3, as indicated. (A) Levels of p53 and p21 proteins were assessed by Western blot analysis of cell lysates; tubulin staining is shown as loading control. Representative examples of Western blot results of 3 to 5 independent experiments are shown. In parallel, activation of GDF15 p53 target gene was assessed by ELISA on supernatants from cell cultures (40 000 cells/mL). Data are mean plus or minus SD of the results from 3 to 5 independent experiments each performed in duplicate (*P < .05, compared with scrambled-transfected and left untreated; **P < .05, compared with scrambled-transfected and treated with Nutlin-3). OPG release in supernatants of the transfected cultures was assessed by ELISA. Results were obtained from 3 (C) or 7 (B) independent experiments, each performed in duplicate. Horizontal bars represent median, upper, and lower edges of box (75th and 25th percentiles); lines extending from box, 10th and 90th percentiles.