Atox1−/− mice (backcrossed eight times to C57Bl/6) were obtained from Mutant Mouse Regional Resource Centers. Atox1−/− mice were originally reported to have phenotypes which show perinatal death (45% of pups) or survive more than one month.20 (link) Our laboratory further backcrossed Atox1−/− mice to C57Bl/6 mice more than ten times, and thus used C57Bl/6 mice as control. “Survivor” mice were intercrossed with more than 10 times and Atox1−/− mice used in the present study survived more than six months (90%). For in vivo experiments, we performed Ang II infusion by osmotic minipumps, blood pressure measurement by the tail cuff method, immunohistochemical and western analysis, real-time PCR, SOD activity assay, vascular O2•− production, vascular reactivity studies by wire myograph, copper measurements by inductively coupled plasma mass spectrometry (ICP-MS), and synchrotron X-ray Fluorescence Microscopy analysis. All studies were approved by the Animal Care and Use Committee of the University of Illinois-Chicago. For in vitro experiments, rat vascular smooth muscle cells (VSMCs) (between passage 7 and 15) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine serum. Using them, we performed nuclear/cytoplasmic fractionation, immunofluorescence, plasmid constructs, transient transfection and reporter assay, and DNA pull down assay.
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