Supplemental Material

This article contains the following supporting material:

• Figure 1 - Disruption of eEF1A-1 in CHO cells by insertional mutagenesis confers resistance to palmitate-induced apoptosis. Wild type and eEF1A-1 null mutant CHO cells were incubated with 500 μM palmitate (Palm), 80 nM staurosporine (Staur), 2 μM actinomycin D (Act D), 20 μM cycloheximde (Cyclo), or 10 μM camptothecin (Camp) for 18 h. Caspase-3 activation was determined using a fluorimetric caspase-3 assay kit. Data expressed as mean ± SEM for five independent experiments, * p<0.05.
• Figure 2 - H9c2 myocyte-like cultures generate ROS and undergo apoptosis and cell death in response to palmitate. (A) Differentiated H9c2 cells were incubated for 5 h with palmitate, followed by 30 min incubation with H₂DCFDA. Mean DCF fluorescence, indicative of relative cellular ROS level, was measured by flow cytometry. (B) Cells were incubated for 24 h with palmitate. Apoptosis was determined by fragment end-labeling (FragEL) and flow cytometry. (C) Cells were incubated as in (B) and cell death was determined by propidium iodide staining and flow cytometry. All data expressed as mean ± SEM for five independent experiments, < p<0.05.
• Figure 3 - Targeted knockdown of eEF1A-1 expression confers palmitate-resistance in H9c2 rat cardiomyoblasts. Wild type H9c2 myoblasts (circles) and H9c2-derived cell lines expressing control siRNA (siRNA1, triangles) or siRNA directed against eEF1A-1 (siRNA2, squares, and siRNA3, diamonds) were incubated for 24 h with increasing concentrations of palmitate. Cell death was determined by propidium iodide staining and flow cytometry.
• Figure 4 - Wild type CHO cells accumulate ROS in response to palmitate. Wild type CHO and mutant cells were incubated for 5 h with 500 μM palmitate, followed by 30 min incubation with H₂DCFDA. Cells were harvested and mean DCF fluorescence, indicative of relative cellular ROS level, was measured by flow cytometry. Data expressed as mean ± SEM for four independent experiments, < p*0.05.
• Figure 5 - Residual band detected in eEF1A-1 null mutant CHO cells decreases in response to palmitate. Wild type CHO cells were incubated with 500 μM palmitate, followed by immunoblotting of whole cell lysates with a monoclonal eEF1A-1 antibody. Inset is a representative blot. Data expressed as mean ± SEM for three independent experiments.
• Figure 6 - Targeted knockdown of eEF1A-1 expression in H9c2 rat cardiomyoblasts prevents induction of eEF1A-1 protein in response to palmitate. Wild type H9c2 myoblasts and H9c2-derived cell lines expressing control siRNA (siRNA1) or siRNA directed against eEF1A-1 (siRNA2 and siRNA3) were incubated for 5 h in the absence or presence of 500 μM palmitate, followed by immunoblotting of whole cell lysates with a monoclonal eEF1A-1 antibody.
• Figure 7 - Disruption of eEF1A-1 in CHO cells by insertional mutagenesis confers resistance to ER stress-induced cell death. Wild type CHO cells and eEF1A-1 null mutant cells were incubated for 48 h with either 2.5 μg/ml tunicaycin or 1 μM thapsigargin. Cell death was determined by propidium iodide staining and flow cytometry.