p53 knock-down up-regulates OPG release in endothelial cultures. HUVECs were transfected with either an EGFP construct or with the indicated siRNA (scr., control scrambled). (A) Efficiency of transfection in each experiment was monitored by flow cytometric and microscopy analyses of EGFP-transfected HUVEC. Representative flow cytometric profiles and microscopy fields (original magnification ×10) of HUVEC cultures transfected with the EGFP-plasmid are shown. (B) Efficiency and specificity of the p53 knock-down were documented by real-time reverse transcription-PCR analyses (qRT-PCR) and Western blot. Results, from amplifications done in triplicate, are expressed as relative RNA levels calculated, after normalization for GAPDH, with respect to control not transfected cultures, which were set to 100. One of 3 Western blot experiments with similar results is shown. Error bars are SD. (C) OPG released in culture supernatants was measured in HUVEC cultures, either not transfected or transfected with the indicated siRNA. Results were obtained from 6 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.