Figure 6. Atox1 is involved in endothelial ROS production in ischemic tissues and ECs stimulated by TNFα in a Cu-dependent and ATP7A-independent manner.

(A) O₂^•- detection probe, dihydroethidium (DHE) was injected into mice at 30 min before sacrifice at day 7, and tissues were stained with CD31. Representative pictures for CD31 staining, DHE fluorescence, and their merged images in ischemic muscles in WT and Atox 1 KO mice (10μm thickness). DHE+/CD31+ double positive cells (yellow) are shown in white arrows. Bars = 20μm. Lower panel shows quantification of number of CD31+ cells and ratio of CD31+/DHE+ cells (n = 3). (B) HUVECs transfected with siRNAs for Atox1, CTR1, ATP7A, control, or treated with either BCS (200uM for 48hrs) or PEG-catalase (500U/ml for 1 hr) were stimulated with TNFα (10ng/ml) for 18hrs. Representative images for DCF fluorescence and DAPI staining (blue, nucleus marker) (left) and quantification of fluorescence intensity (right) (n = 3). *p < 0.05 and ***p < 0.001.