Fig. 5: The BH4 domains of Bcl-2 and Bcl-X_L inhibit pathological TLC-S-induced Ca²⁺ release and necrosis in PACs by inhibiting RyR activity.

a Average traces of Fluo-4AM single-cell Ca²⁺ measurements performed in PACs. The cells were pre-treated for 5 min with either DMSO (vehicle; red) or 50 µM of the peptides: control (blue), the BH4 domain of Bcl-2 (black), and the BH4 domain of Bcl-X_L (pink). Then, TLC-S (200 µM) was added and pathological intracellular Ca²⁺ release was measured for 5 min, in the continuous presence of either vehicle or the peptides. Average traces ± SEM of all performed experiments are shown. b Quantification of the experiments: Analysis of area under the curve per second (AUC/sec) of the TLC-S-induced Ca²⁺ releases, in the presence or absence of the BH4 domains or control peptide. Each data point represents the measurement of an individual cell, and the average values ± SEM are shown (P-value <0.0001). At least three independent experiments were performed per condition (N ≥ 3). For each condition at least 35 cells were recorded. c Quantification of the necrosis assay: Isolated PACs were treated with DMSO (vehicle) or 50 µM of the indicated peptides. 15 min later, TLC-S was added (200 µM final concentration) to induce necrotic cell death. Propidium iodide staining (necrosis indicator) was assessed 2 h after TLC-S addition. The negative control was treated with DMSO (vehicle) only. In each experimental repeat (N = 4) at least 15 images were taken per treatment group (n ≥ 100 cells/treatment/experiment). The percentages of propidium iodide-positive necrotic cells were assessed for each experimental condition. Each data point represents an independent repeat of the experiment, and the average values ± SEM are shown (P-values <0.001)