Fig. 4: Bcl-xL, but not Bcl-xL^K87D, overexpression inhibits IP₃R-mediated Ca²⁺ release in single cells.

Calcium measurements obtained from Fura-2-loaded HeLa (a–c) and COS-7 cells (d–g) transfected with a Bcl-xL (pCMV24-3xFLAG-Bcl-xL-P2A-mCherry; green) or Bcl-xL^K87D-coding vector (pCMV24-3xFLAG-Bcl-xL^K87D-P2A-mCherry; red), or an empty vector (pCMV24-P2A-mCherry; black). ER Ca²⁺ response is elicited by addition of 70 nM (HeLa) or 500 nM (Cos-7) ATP, after addition of 3 mM EGTA to chelate extracellular Ca²⁺. Ionomycin (5 µM) diluted in 250 mM CaCl₂ was added at the end of the experiment (not shown) to trigger a high Ca²⁺ release and confirm all the cells are equally loaded with Fura-2. For each condition, three to five wells were monitored and about 20–30 cells were analyzed by well. a Representative traces of Ca²⁺ release in COS-7 cells. Traces represent mean ± SEM of one representative measurement (one well, about 20–30 cells). Percentage of responding cells (b) and maximal peak amplitude (c) were calculated for each condition. Data represent mean ± SD of four independent experiments (N = 4). Statistically significant differences were determined using a one-way ANOVA (*P < 0.05). Non-responding cells are defined as cells in which fluorescence signal measured after ATP stimulation do not exceed the maximal fluorescence value + SEM measured before stimulation. d Representative traces of Ca²⁺ release in HeLa cells. Traces represent diverse Ca²⁺ release patterns for one single cell. Distribution of typical Ca²⁺ release patterns (e), areas under the curve (f) and amplitudes of the maximal Ca²⁺ peak (g) were calculated from the Ca²⁺ traces of the responding cells. Data represent mean ± SD of four independent experiments (N = 4). Statistically significant differences were determined using a one-way ANOVA (*P < 0.05).