Fig. 6: BIRD-2-triggered cell death is reduced by chelating extracellular Ca²⁺ with EGTA in SU-DHL-4 cells.

a Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 mM EGTA 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. b Quantitative analysis detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 3 mM EGTA. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. Data are represented as the mean ± SEM of five independent experiments. Statistically significant differences were determined with a one-way ANOVA (*p < 0.05, ***p < 0.001). c Single-cell cytosolic Ca²⁺ signals detected in SU-DHL-4 cells using the ratiometric Ca²⁺ indicator Fura-2 AM. The addition of 10 µM BIRD-2 and 3 mM EGTA are indicated by the dotted lines. Representative pseudo-color images before and after BIRD-2/EGTA addition are shown. The pseudo-color gradient at the right of the panel indicates an increasing fluorescence ratio. NS not significant.