Fig. 3: SOCE inhibition with GSK-7975A does not reduce apoptosis induced by 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 3 µM GSK-7975A (blue line). To deplete the ER Ca²⁺ store, 3 mM EGTA and 1 µM TG were added as indicated. After store depletion, Ca²⁺ influx was triggered by adding 10 mM CaCl₂. The curves represent the mean ± SEM of three 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 Dose–response curve of GSK-7975A on SERCA2b ATPase activity (%). The Ca²⁺-dependent ATPase activity was measured at maximal (free [Ca²⁺] 3.16 µM) and submaximal (free [Ca²⁺] 0.316 µM) [Ca²⁺] for different treatments, including vehicle (control) and different [GSK-7975A]. Data were normalized to the values obtained in the control condition at maximal [Ca²⁺], which was set at 100%. Data are represented at the mean ± SEM of three independent experiments. e Single-cell analysis of the ER Ca²⁺ levels in HeLa cells transfected with G-CEPIA1er plasmid. Cells were treated with vehicle (gray curve), 1 µM TG (black curve), or 3 µM GSK-7975A (blue curve) 60 s after the addition of 3 mM EGTA. Data are represented as the mean ± SEM of three independent experiments (n > 100 cells/condition). f 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 3 µM GSK-7975A (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of four independent experiments. The BIRD-2-provoked cytosolic Ca²⁺ rise is quantified by measuring the peak amplitude (∆ F₃₄₀/F₃₈₀), shown in g, and the time constant τ (s), which is shown in h. i 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 µM GSK-7975A 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. j Quantitative analysis of four independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 3 µM GSK-7975A. 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 three independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (**p < 0.01, ****p < 0.0001). NS not significant.