Figure 4

Reduction in stress signaling, inflammation, oxidative stress and ER stress by nSMase inhibition. (A) After palmitate treatment in presence or absence of nSMase inhibitor (GW4869), C2C12 myotubes were harvested in lysis buffer. Upper panel: Levels of phosphorylated JNK, unphosphorylated JNK, IκB, BiP and CHOP were measured using Western blotting. β-actin was used as a loading control. Lower panels: quantification of Western blotting data. (B) Gene expression of IL6 was measured by quantitative real time PCR using β-actin as housekeeping gene control. (C) After treatment, myotubes were loaded with DCFH-DA ROS indicator fluorescent probe to quantify the amount of cellular ROS. (D) Nitric oxide levels in culture medium were quantified using Griess reagent. (E) Myotubes viability was measured by MTT assay after treatment. Data are presented as mean + standard deviation. n = 4, ***P < 0.001, **P < 0.01, *P < 0.05, one way ANOVA with Newman-Keuls post test was performed for statistical analyses.