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. 2013 Oct;183(4):1169–1182. doi: 10.1016/j.ajpath.2013.06.018

Figure 9.

Figure 9

Role of NOX2, NOX4, and Rac1 on S1P-induced ROS production in HLMVECs. A: HLMVECs were transfected with 50 nmol/L scrambled (sc) or Nox2 siRNA for 72 hours, washed with ice-cold PBS, loaded with DCFDA, and then exposed to 1 μmol/L S1P for 30 minutes. ROS production was measured by DCFDA fluorescence. Nox2 siRNA inhibits S1P-stimulated formation of ROS. Original magnification, ×20. B: Quantified data show that Nox2 siRNA inhibits S1P-stimulated formation of ROS. Values for ROS production are means ± SD from three independent experiments. C: HLMVECs were transfected with 50 nmol/L scrambled (sc) or Nox4 siRNA for 72 hours, loaded with DCFDA, and exposed to 1 μmol/L S1P for 30 minutes, as previously described. Nox4 siRNA inhibits S1P-stimulated formation of ROS. D: Quantified data show that Nox4 siRNA inhibits S1P-stimulated formation of ROS. Values for ROS production are means ± SD from three independent experiments. E: Immunoblot demonstrating effective knockdown of expression of NOX2 and NOX4 by siRNA. F: Inhibition of Rac1 with NSC23766 blunts S1P-induced translocation of p47phox to the cell periphery and ROS production. HLMVECs grown on slide chambers were pretreated with 50 μmol/L NSC23766 for 30 minutes, exposed to 1 μmol/L S1P for 30 minutes, washed, fixed, permeabilized, probed with anti-p47phox antibody, and examined by immunofluorescence microscopy. Original magnification, ×60 (oil objective). Exposure of cells to S1P resulted in redistribution of p47phox to the cell periphery, whereas NSC23766 blunted p47phox redistribution. A representative image from one of the three independent experiments is shown. G: HLMVECs were pretreated with 50 μmol/L NSC23766 for 30 minutes and exposed to 1 μmol/L S1P for 30 minutes, and total ROS production was measured by DCFDA fluorescence. Original magnification, ×20. Values for ROS production are means ± SD from three independent experiments. Significantly increased from control untreated cells (sc). ∗P < 0.05, significant decrease of ROS formation compared with control. NO, normoxia; Veh, vehicle.