Alamethicin

BH3 Profiling was performed using the iBH3 plate-based method as previously published (56 (link)). In brief, cell lines were seeded at a density of 1 x 10⁶ cells/mL 24 hours before profiling. Four million cells of each cell line were pelleted at 300 x g for 5 minutes and resuspended in 2mL MEB-P25 (150mM Mannitol, 10mM HEPES-KOH pH 7.5, 150mM KCl, 1mM EGTA, 1mM EDTA, 0.1% BSA, 5 mM Succinate, 0.25% Polaxamer 188[Fisher, MT61161RM]). Cells were permeabilized with digitonin (Sigma, D5628) exposed to BH3 peptides for 60 minutes at 25°C and mitochondrial Cytochrome C release was measured by flow cytometry using a FITC-conjugated antibody (BioLegend, 983502). BH3 peptides were synthesised by New England Peptide using published sequences (100 (link)). The Bcl-XL-selective inhibitor A-1331852 (Selleckchem, S7801), the Bcl-2-selective inihibtor ABT-199 (Selleckchem, S8048) and the MCL-inhibitor AZD5991 (Selleckchem, S8643) were used at a concentration of 10 μM. Results were only deemed valid where cell cytochrome C release in presence of DMSO control was <10% and cytochrome C release in presence of 50ug/mL(25uM) Alamethicin (Enzo, BML-A150-0005) was >90%. Figure represents mean of three independent experiments.

Pyruvate, malate, succinate, glutamate, EGTA, ADP, oligomycin, rotenone, antimycin A, 2,4-dinitrophenol, N-methyl-D-glucamine and carbonylcyanide-p-trifluorometh-oxyphenylhydrazone (FCCP) were purchased from Sigma (St. Louis, MO, USA). Tetraphenylphosphonium chloride was from Fluka (Buchs, Switzerland). Percoll was from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA). Bovine serum albumin (BSA), free from free fatty acids, was from MP Biomedicals (Irvine, CA, USA). Alamethicin was from Enzo (Farmingdale, NY, USA).

Recombinant, mutant perfringolysin O (rPFO) was obtained from Agilent Technologies (XF Plasma Membrane Permeabilizer, XF PMP) and kept as a frozen stock at −20°C. Alamethicin was purchased from Enzo Life Sciences and kept as a 20 mg/mL stock solution in ethanol at −20°C. All other reagents were purchased from Sigma unless otherwise specified.