Figure 5. TLR5-deficient MAECs fails to induce the expression of pro-inflammatory molecules and Nox4 regulates monocyte adhesion and migration in response to flagellin.

MAECs from WT and TLR5 KO were stimulated with flagellin (100 ng/ml) for 2hrs (A,B). (A) Quantification of flagellin-dependent ICAM-1 mRNA expression in MAECs from WT or TLR5 KO mice (N = 3, mean ± SD, **p < 0.01). (B) Quantification of flagellin-induced KC mRNA expression in MAECs from WT or TLR5 KO mice (N = 3, mean ± SD, **p < 0.01). (C) Control or Nox4 siRNA-transfected HAECs were stimulated in the absence or presence of flagellin (100 ng/ml). Pre-stained U937 cells with BCECF-AM (5 μg/ml) were incubated with HAECs for 30 min. The number of adherent U937 cells was determined (N = 3, mean ± SD, ***p < 0.001). (D) HAECs were transfected control or Nox4 siRNA in lower chamber and then stimulated with flagellin. U937 cells in upper chamber were allowed to migrate on fibronectin-coated Transwell plates for 4 hr. Trans-migrated U937 cells were counted under an inverted fluorescence microscope (N = 3, mean ± SD, ***p < 0.001). (E) Monocyte adhesion assay in flow condition. HAECs transfected control siRNA or Nox4 siRNA were exposed to laminar shear stress (15 dyn/cm²) or oscillatory shear stress (±5 dyn/cm²) with or without flagellin (100 ng/ml) for 24 hrs using the cone-and-plate apparatus. Pre-stained U937 cells with BCECF-AM (5 μg/ml) were incubated with HAECs for 30 min. The number of adherent U937 cells was determined (N = 3, mean ± SD, **p < 0.01, ***p < 0.001).