Figure 2. Nox4 is required for flagellin-induced ROS generation.

(A) Wild type (WT), Nox1 knockout (KO), and Nox4 KO MAECs were 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) WT, Nox1 KO, and Nox4 KO MAECs were incubated for 10min with flagellin (100 ng/ml), H₂O₂ was monitored by a confocal microscopic analysis of PO-1 fluorescence (N = 3, mean ± SD, **p < 0.01). (C) HAECs were transiently transfected with control or Nox4 siRNA, and then incubated 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). (D) Nox4 protein expression was analyzed by immunoblotting with an antibody against Nox4. (E) HAECs were pretreated with PEG-catalase (50 U/ml) or DPI (20 μM) or VAS2870 (5 μM) for 30 min, 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).