Figure 7
The effect of AMPK knockdown on the zileuton action. C2C12 myotubes were transfected with negative control siRNA or AMPK siRNA (20 nM), treated with 50 μM zileuton for 1 h, and then with PA (750 μM) for 12 h (qPCR) or 24 h (western blot, ELISA, ROS, and glucose uptake). The effect of AMPK knockdown was determined by western blotting (a). The effects of AMPK siRNA on Akt/serIRS-1 phosphorylation, and AMPK/ACC phosphorylation (b) and ER stress markers were determined by western blotting (c) and qPCR (d). The expression of 5-LO was determined by qPCR (d). Cellular TNF-α and IL-6 mRNA levels and secreted TNF-α and IL-6 levels were determined by qPCR and ELISA (e). ROS production was assessed using AmplexRed (f). Glucose uptake was determined by 2-NBDG in the presence or absence of insulin (1 μg/ml) for 30 min (g). LTB4 production was measured by ELISA kits (h). The results of the western blotting are shown as the representative of three independent experiments (b and c), and other results are expressed as the means ± SDs of three experiments performed in triplicate. # P < 0.05 vs. non-treated controls, *P < 0.05 vs. PA alone, **P < 0.05 vs. control siRNA of PA plus zileuton, & P < 0.05 vs. (−) insulin, ns; not significant.
