Figure 1

Effects of zileuton on PA-induced ER stress and oxidative stress. The mRNA expressions of 5-LO, BLT1, CysLT1 and CysLT2 in C2C12 myotubes were determined by qPCR with or without PA treatment (750 μM for 12 h) (a, upper). 5-LO and BLT1 protein levels were determined by western blotting with or without PA treatment (a, lower). LTB4 production was determined by LTB4 kits, with or without PA treatment (750 μM for 24 h) (a). The mRNA expressions of CysLT1 and 2 receptors were determined by qPCR with or without PA treatment (b). C2C12 myotubes were pre-exposed to different concentrations of zileuton (1-50 μM) for 1 h and then treated with PA (750 μM) in the presence of zileuton for 12 h (qPCR) or 24 h (western blot, LTB4/CysLTs ELISA and ROS). The expression levels of ER stress markers were then determined by western blotting (c) and qPCR (d). ROS production was assessed using AmplexRed (e), and LTB4 and CysLTs production was determined by LTB4 and CysLTs ELISA kits, respectively (f). Cell viability was determined after 24 h PA treatment by MTT assay (g, upper) and LDH release measurement (g, lower). The results of the western blotting are shown as the representative of three independent experiments (a and c), and other results are expressed as the means ± SDs of three experiments performed in triplicate. ^# P < 0.05, ^## P < 0.01 vs. non-treated controls, *P < 0.05 vs. PA alone. ns; not significant.