Oligomycin b

DiBAC₄(3) was obtained from Thermo Fisher Scientific. Rotenone, antimycin, oligomycin B and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (all from Sigma-Aldrich, Dorset, UK) were dissolved in DMSO as 1, 1, 6 and 10 mM stock solutions, respectively. K⁺ channels inhibitors, glibenclamide (Sigma-Aldrich), penitrem A (Alomone labs, Jerusalem, Israel), tram34 (Sigma-Aldrich), apamin (Tocris Bioscience, Abingdon, UK) and XE991 (Tocris Bioscience) were also prepared in DMSO as 10, 1, 10, 1 and 10 mM stock solutions, respectively. Other chemicals were obtained from Sigma-Aldrich or VWR (Leicestershire, UK).

Autobioluminescent T47D or HepG2 cells were seeded at ~1 × 10⁴ or 2.5 × 10⁴ cells/well, respectively, in flat-bottom black 96-well plates and incubated under standard conditions (37 °C, 5% CO₂). After overnight incubation, medium was removed from the cells and replaced with fresh medium containing Cytarabine, Methotrexate, or Oligomycin B (Sigma-Aldrich, Saint Louis, MO, USA) at concentrations ranging from 1 nM to 10 µM or with 0.1% Dimethyl sulfoxide (DMSO) as a control. Test compounds were prepared in DMSO and the final concentration of DMSO was 0.1% in all wells. Each compound was tested in triplicate plates and each concentration was tested in triplicate wells per plate. After dosing, cells were incubated under standard conditions for 24 h before they were interrogated in a Synergy2 plate reader (Bio-Tek, Winooski, VT, USA) using the CellTiter-Glo assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol.