Figure 3: Nox4 is required for flagellin-induced NF-kB activation in HAECs.

(A) HAECs were transfected with control or TLR5 siRNA, and stimulated for 10 min with flagellin (100 ng/ml). H₂O₂ was monitored by a confocal microscopic analysis of DCF fluorescence (N = 3, mean ± SD, ***p < 0.001). (B) TLR5 protein expression was analyzed by immunoblotting with an antibody against TLR5. (C) HAECs were stimulated with flagellin (100 ng/ml), rFliC (100 ng/ml), or ΔrFliC (100 ng/ml) for 10 min. H₂O₂ was monitored by confocal microscopic analysis of DCF fluorescence. (N = 3, mean ± SD, ***p < 0.001). (D) Wild type and TLR5 knockout MAECs were stimulated for 10 min with flagellin (100 ng/ml). H₂O₂ was monitored by confocal microscopic analysis of DCF fluorescence. (N = 3, mean ± SD, ***p < 0.001). (E) Wild type and MyD88 knockout MAECs were stimulated for 10 min with flagellin (100 ng/ml). H₂O₂ was monitored by confocal microscopic analysis of DCF fluorescence (N = 3, mean ± SD, ***p < 0.001). (F) HAECs were transfected with control siRNA (non-targeting siRNA), Nox4 siRNA, or TLR5 siRNA, stimulated with flagellin (100 ng/ml) for 1 hr and stained with antibody to p65 (red) and DAPI (blue). A representative image is shown. Scale bar: 20 μm. (G) Nuclear translocated p65 was quantified using an image J program (N = 3, mean ± SD, ***Comparison with non-stimulated control group, p < 0.001, ^###Comparison with flagellin stimulated control group, p < 0.001).