A 1331852

Two mg of isolated mitochondria were suspended in a total volume of 1 ml consisting KCl buffer, 1 mM Malic acid (Sigma-Aldrich), 7 mM Pyruvate (Sigma-Aldrich), and 50 nM Calcium Green 5N (Invitrogen) in a quartz cuvette, which was placed inside the fluorimeter (PTI QuantaMaster 800, Horiba Scientific). Calcium uptake was measured by fluorescent emission of Calcium Green 5N. Simultaneously, mitochondrial swelling was measured by transmittance light. Each BH3 mimetic was used at concentrations: 0.5, 1, 10, 50, 100 nM, and 1 µM. Final BH3 mimetics concentrations used throughout the study were 200 nM ABT-199 (Selleckchem.com, S8048), 100 nM A-1331852 (abcam, ab218112), 100 nM ABT-737 (EMD Millipore Sigma, 197333), 10 nM S63845 (Selleckchem.com, S8383), 10 nM Obatoclax (Selleckchem, GX15-070). For some experiments ADP (300 uM) (Sigma-Aldrich, A2754) and/or CsA (2 uM) (Sigma-Aldrich, 30024) were used to desensitize the MPTP. CaCl₂ (20 μM, 40 μM, and 80 µM) (Sigma-Adrich, C4901) was added into this system in succession until MPTP opening occurred, indicated by an upturn in Calcium Green 5N fluorescence, or when mitochondria were saturated with Ca²⁺ and were no longer able to take up further additions of CaCl₂, indicated by a stair-stacking in the Ca²⁺ uptake graphs.

Specific BclxL inhibitors were purchased from Abcam (inhibitor #1, A-1155463; inhibitor #2, A-1331852) and added to the cultures at a concentration of 1 µM (based on the pilot dose–response experiment).