Fig. 3— Amifostine (Amif) inhibits reactive oxygen species (ROS) production induced by lipopolysaccharide (LPS). Human pulmonary artery endothelial cells were pre-treated with a–c) vehicle, d–f) N-acetylcysteine (NAC; 5 mM, 1 h), or g–i) WR-1065 (4 mM, 30 min). This was followed by 6 h LPS stimulation of a, d and g) 100 ng·mL−1, b, e and h) 200 ng·mL−1, or c, f and i) 500 ng·mL−1. ROS was detected by carboxy-H2DCFDA and carboxy-H2DCFDA-5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescin diacetate fluorescent assay, as described in the Materials and Methods. The results are representative of the entire cell monolayer and have been reproduced in three independent experiments. j) Statistical analysis of the effect of NAC and Amif on LPS-induced ROS production. AU: arbitrary units. *: p<0.05, compared with 500 ng·mL−1 LPS treated endothelial cells; #: p<0.005, compared with NAC or amifostine pre-treated EC.