Fig. 7: Wild-type Bcl-xL, but not Bcl-xL^K87D, protects HeLa cells against IP₃R/Ca²⁺-driven cell death using staurosporine.

a, b Ca²⁺ measurements in Fura-2-loaded wild-type HeLa (black) and HeLa-3KO cells (blue). Cells were exposed to 0.5 µM staurosporine (STS), after addition of 3 mM EGTA to chelate extracellular Ca²⁺ (not shown). Representative traces of Ca²⁺ release are shown (a), along with areas under the curve (b) calculated for one hour following STS addition. For each experiment (N = 2), two wells were monitored per condition and about 20–30 cells were analyzed by well. Each trace and each point represent one cell. Statistically significant differences were determined using a t test (*P < 0.05). c, d Wild-type HeLa and HeLa-3KO cells were treated with 0.5 µM STS or vehicle (DMSO) for 6 h. The samples were analyzed via western blot (IB: immunoblot). Representative western blots assessing uncleaved (top band) and cleaved PARP (lower band) as well as vinculin (c). The immunoreactive bands from independent experiments, using each time freshly prepared cell lysates, were quantified (d) and PARP cleavage was calculated as the ratio of cleaved PARP over total PARP. The data are plotted as mean ± SD (N = 6). Statistically significant differences between the “+STS” conditions were determined using a t test (paired, two-tailed, *P < 0.05). e, f Ca²⁺ signals were measured in Fura-2-loaded wild type HeLa expressing empty vector (pCMV24-P2A-mCherry; black), Bcl-xL (pCMV24-3xFLAG-Bcl-xL-P2A-mCherry; green) or Bcl-xL^K87D (pCMV24-3xFLAG-Bcl-xL^K87D-P2A-mCherry; red). ER Ca²⁺ release is triggered as in a. The traces represent the average response of all cells ± SEM in one well containing about 20 cells (e). The individual Ca²⁺ traces are shown in Fig. S7a. For each condition, 1 to 2 independent wells obtained from 4 different transfections were monitored. The areas under the curve were calculated for all individual cell during the 45 min following STS addition (f). Data are represented as mean of wells ± SD (N = 5 to 7), each data point represents one well. Statistically significant differences were determined using a one-way ANOVA (*P < 0.05). Another graphical representation is shown in Fig. S7b. g–j Wild type HeLa (g, h) and HeLa-3KO cells (i, j) transiently overexpressing 3xFLAG-Bcl-xL or 3xFLAG-Bcl-xL^K87D were treated with 0.5 µM STS or vehicle (DMSO) for 6 h. The samples were analyzed via western blot. Representative western blots assessing uncleaved and cleaved PARP as well as total Bcl-xL (endogenous + overexpressed) and β-actin (g, i). The vertical line in panel g indicates that two different parts of the same gel and exposure time were merged together. The original uncropped picture is shown in Fig. S7c. The immunoreactive bands from independent experiments, using each time independently transfected and treated cells and freshly prepared cell lysates, were quantified (h, j) and PARP cleavage was calculated like in d. The data are plotted as mean ± SD (N = 5). Statistically significant differences between the “+STS” conditions were determined using a one-way ANOVA (*P < 0.05). k, l Wild-type HeLa and HeLa-3KO cells transiently overexpressing 3xFLAG-Bcl-xL or 3xFLAG-Bcl-xL^K87D were treated with 25 µM venetoclax or vehicle (DMSO) for 24 h. The samples were analyzed via western blot. Representative western blots assessing uncleaved (top band) and cleaved PARP (lower band), as well as overexpressed Bcl-xL (FLAG) and β-actin (k). The immunoreactive bands from independent experiments, using each time independently transfected and treated cells and freshly prepared cell lysates, were quantified and PARP cleavage was calculated as in d (l). The data are plotted as mean ± SD (N = 5). Statistically significant differences were determined using a one-way ANOVA (*P < 0.05).