Nutlin-3 counteracts the induction of OPG by TNF-α. HUVECs were left untreated or treated with TNF-α in the presence or absence of the indicated concentrations of Nutlin-3. OPG levels in culture supernatants were assessed by ELISA after 8 (A) and 24 (B) hours of treatment. Data are expressed as mean plus or minus SD of results from 4 independent experiments, each performed in duplicate (*P < .05, compared with TNF-α alone). (C) The relative levels of OPG mRNA, and for comparison, also of p21 mRNA were determined by real-time reverse transcription-PCR. Results from amplifications done in triplicate are expressed as relative RNA levels calculated, after normalization for GAPDH, with respect to control untreated cultures, which were set to 1 (*P < .05).