Figure 4. ER stress-generated LTC₄ triggers NOX4-mediated ROS accumulation in WISH cells.

(a,b) ROS detection using dichloro-dihydrofluorescein diacetate (DCFH-DA, hydrolysed and oxidized by ROS to dichlorofluorescein (DCF)) in cells transfected with control siRNA or Mgst2 siRNA, and then treated with vehicle or BfA. Bar, 100 μm. n=4, **P<0.01. (c,d) ROS detection in cells treated with vehicle or BfA in the absence or presence of LTC₄ receptor antagonists or the MRP1 transporter inhibitor reversan. Treatment with reversan or the receptor antagonists alone did not induce ROS. Bar, 100 μm. n=4, *P<0.02; ***P<0.001. (e,f) ROS detection and quantification in cells transfected with control siRNA or Nox4 siRNA, and then treated with vehicle or BfA. Bar, 100 μm. n=5, ***P<0.001. (g) Immunoblot of NOX4 in extracts of cells transfected with control siRNA or Nox4 siRNA, and then treated with vehicle or BfA. (h) Immunostain of NOX4 in cells treated with vehicle or BfA. Merge of Hoechst and NOX4 is shown. Bar, 5 μm. (i) Per cent nuclear localization of NOX4 as determined by analysis of confocal images shown in e. n=8, ***P<0.0001. (j) Immunoblot of NOX4 and NOX2 in extracts of cells treated with the indicated LTC₄ receptor antagonists together with vehicle (−) or BfA (+). Blots g and j are representatives of three replicates. Values in b,d,f and i represent the mean±s.d. Statistical significance was determined using one-way ANOVA.