Figure 6

A. Immunoblot analysis of A549 cells treated with EM011 for 0, 24, 48 and 72 hrs. After the indicated times, cells were lysed and total protein was extracted, separated by SDS-PAGE, electrotransferred onto polyvinylidene difluoride membrane, and subjected to immunoblotting with the indicated primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. β-actin was used as a loading control. B. Knock-down of survivin using survivin siRNA enhances the apoptotic response of A549 cells to EM011 treatment. A549 cells were transfected with a plasmid encoding survivin siRNA and were then subjected to EM011 exposure for 0, 24, 48 and 72 hrs. An empty vector control was used in the experiments. Panel B shows three-dimensional FACS profile of survivin siRNA-transfected (T) and empty vector transfected (EV) cells for 0, 24, 48 and 72 hrs. C. Western blot analysis of the expression of survivin and β-actin in A549 cells transfected with control or survivin siRNAs. D. Quantitation of the percent sub-G1 population for control siRNA and survivin siRNA transfected cells. Values represent averages and error bars show SD (*, p < 0.05). E. Overexpression of survivin renders A549 cells resistant to EM011-induced apoptosis. A549 cells were transfected with a survivin encoding plasmid construct to ectopically overexpress survivin. Panel E shows cell-cycle profiles of survivin-transfected cells along with empty vector-transfected controls. F. Western blot analysis of the expression of survivin and β-actin in A549 cells transfected with control or survivin plasmid. G. Quantitation of the percent sub-G1 population for the survivin-transfected and empty vector-transfected controls. Values represent averages and error bars show SD (^#, not significant; *, p < 0.05). H. Quantitation of the time-dependent increase in caspase-3 activity upon EM011 treatment. Cells were treated with 25 μM EM011 for 0, 24, 48, and 72 hrs, and caspase-3 activity was analyzed using the luminogenic substrate Z-DEVD-aminoluciferin. The values and error bars shown in the graph represent the averages and standard deviations, respectively, of three independent experiments; (p < 0.05).