Figure 6

Analysis of caspase-3 activation and mitochondria involvement during the cell death induced by anti-GD2 antibodies. Enzymatic activity of caspase-3 in control (untreated) EL-4 cells, or treated with anti-GD2 mAbs 14G2a (5 μg/ml), or staurosporine (50 nM) for 24 h is shown in (A). Mean ± S.E. of three separate experiments is shown. The statistical analysis was performed using two way analysis of variance method. There was a statistically significant differences between groups (P ≤ 0.001), *** P < 0.001 as determined by multiple comparisons of experimental versus control groups using Dunnett's post-hoc analysis. Effect of Pan-caspase inhibitor Z-VAD-FMK (10 μM) on cell death induced by anti-GD2 mAbs 14G2a (5 μg/ml) and Staurosporine (50 nM) after 24 h of incubation with EL-4 cells is shown in (B). Statistical analysis was performed using Mann–Whitney rank sum test, the differences between control and pan-caspase inhibitor groups were statistically significant (*, P < 0.05; ***, P < 0.001). Effect of anti-GD2 mAbs 14G2a (5 μg/ml) and staurosporine (50 nM) on ΔΨm of AVD-positive and 7AAD-negative populations of EL-4 cells. Representative density plots of flow cytometry analysis of mitochonodrial potential (MPT) measured by using JC-1 probe (2 μg/ml) in intact versus the cells incubated with anti-GD2 mAbs 14G2a (5 μg/ml) or staurosporine (500 nM) for 2 h is shown. (C). Representative density plots of MPT measured by using DioC₆(3) probe (20 nM) in intact and cells incubated with anti-GD2 mAbs 14G2a (5 μg/ml) or staurosporine (500 nM) for 2 h is shown (D).