Figure 4.

The influence of silencing of lncRNA XIST on the migration and tube formation and the expression of Itgα5, KLF4, three CAMs, phosphorylation of (p)-NF-κB, and TJPs of bEnd3 cells under OGD/R conditions

(A) Representative images of western blot for the expression of Itgα5, KLF4, three CAMs (E-selectin, VCAM-1, and ICAM-1), p-NF-κB, and TJPs (Claudin-5 and ZO-1) in bEnd3 cells transfected with the negative control siRNA (si-Ctl) or siRNA-lncRNA XIST (si-XIST) at 24 h restoration from OGD. The NO-OGD/R cells served as a control. (B−I) Bar graphs show the quantitative analyses of western blots as ratios of Itgα5/β-actin (B), KLF4/β-actin (C), E-selectin/β-actin (D), VCAM-1/β-actin (E), ICAM-1/β-actin (F), p-NF-κB/total NF-κB (G), Claudin-5/β-actin (H), and ZO-1/β-actin (I) (n = 4 per experimental group). Note that the expressions of Itgα5, KLF4, and TJPs (Claudin-5 and ZO-1) in the si-XIST-treated bEnd3 cells were markedly reduced, but the expressions of three CAMs including E-selectin, VCAM-1, and ICAM-1 and the p-NF-κB were significantly elevated relative to the si-Ctl-treated group at 24 h restoration from OGD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (J) Representative images of cell migration and capillary tube formation of bEnd3 cells transfected with si-Ctl or si-XIST after OGD/R treatment. Scale bar, 400 μm for migration; 500 μm for tube formation. (K) Quantification of cell migration of bEnd3 cells. The relative migration distance was quantified using ImageJ software and expressed as the ratio to the values of the NO-OGD/R cells (control). (L) Quantification of tube formation of bEnd3 cells. Data represent the mean ± standard deviation and are analyzed by one-way ANOVA (n = 4 per experimental group). Note that the migration and capillary-like tube formation of the si-XIST-treated bEnd3 cells were both significantly induced after OGD/R relative to the NO-OGD/R control, but this effect was significantly inhibited by silencing of lncRNA XIST in bEnd3 cells. ∗p < 0.05 and ∗∗p < 0.01.