Fig. 6

The influence of knockdown of KLF4 on the expression of three cell adhesion molecules, phosphorylated NF-κB, tight junction proteins, and TNF-α in bEnd3 cells under OGD/R conditions. a Representative images of western blot for the expression of KLF4 in the bEnd3 cells in the negative control siRNA (siRNA-Ctl) or three different KLF4-specific siRNA (siRNA-KLF4)-treated group. Note that three different specific siRNA against the murine KLF4 all reduced the expression of KLF4 in bEnd3 cells compared to that of siRNA-Ctl-treated bEnd3 cells, respectively, and siRNA-KLF4-2 can target the KLF4 with the highest efficiency. b Representative images of western blot for the expression of KLF4, E-selectin, VCAM-1, ICAM-1, p-NF-κB, Claudin-5, and ZO-1 in the bEnd3 cells of siRNA-Ctl and siRNA-KLF4-2 group at 12 h restoration of OGD or NO-OGD/R. c–i Bar graphs show the quantitative analyses of western blots as ratios of KLF4/β-actin (c), E-selectin/β-actin (d), VCAM-1/β-actin (e), ICAM-1/β-actin (f), phosphorylated NF-κB/total NF-κB (g), Claudin-5/β-actin (h), and ZO-1/β-actin (i) (n = 5 per experimental group). j The mRNA level of TNF-α was determined by qPCR in the BECs of siRNA-Ctl and siRNA-KLF4-2 group at 12 h restoration of OGD or NO-OGD/R (n = 4 per experimental group). NO-OGD/R siRNA-Ctl-treated cells served as control. Data represent mean ± standard deviation and were analyzed by two-way ANOVA. Note that the expressions of three cell adhesion molecules including E-selectin, VCAM-1, and ICAM-1 and the phosphorylation of NF-κB from the siRNA-Ctl-treated bEnd3 cells were significantly induced in response to OGD/R, but the expressions of tight junction proteins (Claudin-5 and ZO-1) markedly decreased compared to that of the controls. Furthermore, OGD/R-induced expression of three cell adhesion molecules and the phosphorylation of NF-κB were enhanced by diminishing the levels of KLF4 in the bEnd3 cells; likewise, the decreased expression of tight junction proteins caused by OGD/R were further augmented by silencing the levels of KLF4 in the bEnd3 cells. OGD/R significantly increased the mRNA expression of TNF-α in the siRNA-Ctl-treated bEnd3 cells, but siRNA-mediated knockdown of KLF4 showed no notable effects on the expression of TNF-α following OGD/R. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant