Fig. 7.

GPx7 protects endothelium against Hcy-induced ER oxidative stress. (A) Lysates prepared from the thoracic aortas of normal (Ctrl) or HHcy mice were subjected to immunoblotting and statistical analysis for NRF2 and GPx7 expression. Representative blots were shown. Data were represented as mean ± SEM from eight biological replicates, *p < 0.05 via two-tailed Student's t-test. (B) Immunofluorescence analysis of GPx7 (red, Ex = 568 nm) from the thoracic aortas of Ctrl and HHcy mice with rabbit IgG as a negative control. The aortic intima is indicated by white dashed line. BF, bright field. Scale bars, 50 µm. (C) HUVECs transduced with lentivirus expressing GFP or HA-tagged GPx7 (HA-GPx7) for more than 72 h were treated without or with 200 μM Hcy for 4 h and subjected to immunoblotting. The expression of BiP and p-eIF2α/eIF2α was analyzed. Representative blots were shown. (D) HUVECs lentivirus expressing GFP or HA-tagged GPx7 (HA-GPx7) for more than 72 h were treated without or with different concentration of Hcy for 1 h, and H₂O₂ levels were measured as in Fig. 2C. (E) HUVECs transduced with lentiviral luciferase (Luc) or HA-GPx7 were treated without or with 200 μM Hcy for 4 h. Cells were analyzed by immunofluorescence as in Fig. 5C. Scale bars, 20 µm. The relative fluorescence intensities from dimedone signals were quantified. (C–E) Data were represented as mean ± SEM from three independent experiments, **p < 0.01 via two-way ANOVA, Tukey's multiple comparisons test. (F) Ex vivo aortic ring assays. Mouse thoracic aortas were transduced with adenovirus vector expressing GFP or HA-GPx7 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 and BiP was shown in Fig. S11A, B. (G) In vivo external carotid artery injection assays with adenovirus vector expressing GFP or HA-GPx7 were carried out as in Fig. 5E. Representative blots were shown. The statistical analysis of BiP was shown in Fig. S11C.