Figure 6. Effect of 4E-BP1 siRNA on Rapamycin-Mediated Protection from IL-13-mediated Potentiation of Oxidative Stress-Induced Cell Death.

MN9D cells were transfected with control siRNA or 4E-BP1 siRNA as described in Methods. (A) and (B) Transfected MN9D cells grown in 96 well dishes were untreated or treated with 1, 5 or 10 ng/ml IL-13 alone, in the presence of 80 μM H₂O₂, 2.5 μM tBOOH, 100 nM rapamycin or a combination as indicated. Cell survival was measured after 24 hr with the MTT assay. Results are presented as the fold change in cell death as compared to H₂O₂ or tBOOH alone. The experiments were done in quadruplicate and repeated 6 times. * p < 0.05; ** p < 0.01; *** p < 0.001 relative to no rapamycin. (C) Cell lysates were prepared and equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho and total 4E-BP1. A representative Western blot presenting two independent experiments is shown. Similar results were obtained in 6 independent experiments.