Fig. 5.

Knockdown of Ero1α ameliorates Hcy-induced ER oxidative stress in endothelium. (A) HUVECs transduced with lentiviral control shRNA (shCtrl) or shEro1α for more than 72 h were treated with 200 μM Hcy for an additional 4 h. The protein levels of Ero1α, BiP, XBP1s/XBP1u, p-eIF2α/eIF2α, ICAM-1, and VCAM-1 were determined by immunoblotting and statistical analysis. Representative blots were shown. (B) HUVECs transduced with lentiviral shCtrl or shEro1α for more than 72 h were treated without or with different concentrations of Hcy for 1 h, and H₂O₂ levels were measured as in Fig. 2C. (C) HUVECs transduced with lentiviral shCtrl or shEro1α were treated without or with 200 μM Hcy for 4 h. Sulfenylated proteins (green, Ex = 568 nm) and the ER marker PDI (red, Ex = 647 nm) were analyzed by immunofluorescence. Nuclei were counterstained with Hoechst stain (blue, Ex = 405 nm). Scale bars, 20 µm. The relative fluorescence intensities from dimedone signals were quantified. (A–C) Data were represented as mean ± SEM from three independent experiments, *p < 0.05 and **p < 0.01 via two-way ANOVA, Tukey's multiple comparisons test. (D) Ex vivo aortic ring assays. Mouse thoracic aortas were transduced with adenovirus vector expressing shCtrl or shEro1α for 48 h, followed by treatment without or with 200 μM Hcy for 4 h and immunoblotting analysis. Representative blots were shown. The statistical analysis of ICAM-1, BiP, and Ero1α expression was shown in Fig. S6A–C. (E) In vivo external carotid artery injection assays were performed as described in Methods. Mice were locally injected in the external carotid arteries with adenovirus vector expressing shCtrl or shEro1α for 72 h, then injected with saline or Hcy through the tail vein. After 1 h the carotid arteries were harvested for immunoblotting analysis. Representative blots were shown. The statistical analysis of ICAM-1, BiP, and Ero1α expression was shown in Fig. S6D–F.