Vascular Reactivity and Endothelial Cell Extraction Protocol

Provinols™ was obtained from Société Française des Distilleries (Vallon Pont d'Arc, France) and delphinidin was purchased from Extrasynthèse (Genay, France). The university of Angers ethical committee approved the present protocol. All animal studies were carried out using approved institutional protocols and were conformed the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Methods for vascular reactivity performed in mice [26] (link), [27] (link) and endothelial cells extraction and culture were set up as previously described [19] (link). Methods for RNA interference and transient transfection to silence ERα were adapted from Agouni et al.[26] (link). NO and O₂⁻ spin trapping and electronic paramagnetic resonance (EPR) studies and Western blotting were conducted as previously described [26] (link). Binding assay was performed by CEREP (Paris, France) using fluorescence polarization methods in human recombinant Sf9. Delphinidin was docked on ERα using default settings of the GOLD4.0 program [28] (link). Additional details of the methods used are provided in the Supplemental Data file (Methods S1).