Fig. 1. DPB162-AE reduces the cytotoxic effects of BIRD-2 in SU-DHL-4 cells.

a Analysis of Ca²⁺ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca²⁺ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 10 µM DPB162-AE (blue line). To deplete the ER Ca²⁺ store, 3 mM EGTA and 1 µM thapsigargin (TG) were added as indicated. After store depletion, Ca²⁺ influx was triggered by adding 10 mM CaCl₂. The curves represent the mean ± SEM of four independent experiments. Quantification of the Ca²⁺ influx is provided as the peak amplitude (∆ F₃₄₀/F₃₈₀) in b, whereas the TG-releasable Ca²⁺ is quantified as the AUC (F₃₄₀/F₃₈₀ × s) in c. d Analysis of the cytosolic Ca²⁺ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 10 µM DPB162-AE (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of five independent experiments. The BIRD-2-provoked cytosolic Ca²⁺ rise is quantified by measuring the peak amplitude (∆ F₃₄₀/F₃₈₀), shown in e, and the time constant τ (s), which is shown in f. g 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 10 µM DPB162-AE 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. h Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 10 µM DPB162-AE. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least four independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). NS not significant