Figure 5.

Palmitate-induced ROS activate an ER stress response and induce eEF1A-1 expression and cell death in cardiomyoblasts. (A) Myoblasts were incubated for 1 h with ethanol (vehicle control) or 200 μM α-tocopherol, followed by 5 h with 500 μM palmitate. Expression of ER stress response proteins (GRP78 and CHOP-10) and eEF1A-1 were detected in whole cell lysates by immunoblotting. Cells incubated for 5 h with 2.5 μg/ml tunicamycin were included as a positive control. Relative densitometry values for the representative blots shown are given below each band. Open arrow indicates a nonspecific protein band. (B) Cells were incubated as described in A, followed by incubation for 30 min with H₂DCFDA. Mean DCF fluorescence, indicative of relative cellular ROS level, was measured by flow cytometry. (C) Cells preincubated as described in A were incubated for 24 h with or without 500 μM palmitate. Cell death was determined by propidium iodide staining and flow cytometry. (D) Wild-type (closed symbols) and eEF1A-1 null mutant (open symbols) CHO cells were incubated with 500 μM palmitate. GRP78 protein levels in whole cell lysates were detected by immunoblotting. Insets are representative blots. (E) Wild-type H9c2 myoblast and stable H9c2-derived cell lines expressing control siRNA (siRNA1) or siRNA directed against eEF1A-1 (siRNA2 and siRNA3) were incubated for 48 h with either 2.5 μg/ml tunicamycin or 1 μM thapsigargin. Cell death was determined by propidium iodide staining and flow cytometry. For B-E, data expressed as mean ± SEM for at least three independent experiments, ^* p < 0.05.