Z vad ome fmk

Approximately 5 × 10⁴ mammalian cells were seeded onto glass coverslips (30% confluency) the day before intoxication with 5 ng/ml recombinant typhoid toxin for 2 h at 37 °C. Cells were washed and further incubated, typically 24 h, in complete growth media before phenotypic analysis. Drugs were incubated with cells as indicated in the text or 1 h before intoxication when used in combination with the typhoid toxin and remained present throughout experiments. 20 μM Aphidicolin (A0781, Sigma-Aldrich), 1 μM Killer-TRAIL (ALX-201-073-CO20, Enzo), 100 nM staurosporine (SM97-1 Cambridge Bioscience), 30 μM Z-VAD-OMe-FMK (Selleckchem), 10 μM iATM KU55933, 15 μM iATR NU6072 (Tocris).

BH3 profiling was carried out as previously described.^{34 (link)} In brief, the cells were permeabilised with digitonin (0.002 %) in DTEB buffer (10 mM HEPES, 135 mM Trehalose, 20 μM EDTA, 20 μM EGTA, 5 mM succinic acid, 0.1 % BSA, 50 mM potassium chloride, pH 7.5) containing oligomycin (10 μg/ml) and incubated for 2 h with varying concentrations of BH3 peptides. The loss of mitochondrial membrane potential (ϕm) was measured by observing the loss of TMRE (200 nM) using an Attune NxT flow cytometer (ThermoFisher Scientific, Paisley, UK). The extent of apoptosis in cells following different treatments was quantified by FACS following staining of the cells with Annexin V-FITC and propidium iodide to measure phosphatidylserine externalisation, as previously described.^{33 (link)} For monitoring BAK activation, the cells pre-treated with Z-VAD(OMe).fmk (Selleck Chemicals) for 30 min were exposed to the indicated drugs, fixed in 1 % (w/v) paraformaldehyde and stained with conformation-specific AP-1 BAK antibody (1 μg/ml), corresponding fluorophore- conjugated secondary antibody and quantified by FACS.