Figure 5

Lipotoxicity causes oxidative stress, endoplasmic reticulum stress and ceramide synthesis in myotubes. (A) Post lipotoxicity, myotubes were loaded with DCFH-DA ROS indicator probe and amount of ROS was quantified (** P < 0.01). (B) Nitric oxide levels in culture medium was quantified using Griess reagent (*** P < 0.001). (C) Expression of genes involved in oxidative stress, ER stress and ceramide synthesis in myotubes cultured under lipotoxic condition were quantified by real time RT-PCR and expressed as fold levels of control (Broken line) (* P < 0.05, ** P < 0.01, *** P < 0.001). (D) After chronic lipotoxic conditions, myotubes were harvested for CHOP level measurement by Western blotting. Beta actin was used as a loading control. (E) For estimation of cytoplasmic calcium levels, myotubes cultured under control or lipotoxic condition were loaded with Fluo-4 AM calcium indicator dye and the change in fluorescence was estimated as described in the Methods.