V7 seahorse tissue culture plates

The bioenergetic function of cells in response to drug treatments was determined using a Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Cells were seeded in specialized V7 Seahorse tissue culture plates (Agilent, 102601-100) for 24 hours. Cells were then treated with the indicated concentrations of BAS-2 for further 24 hours. One hour before the experiment, cells were washed and changed to XF Base medium (Agilent, 10353-100) adjusted to pH 7.4. For oxidative phosphorylation experiments, medium was supplemented with pyruvate (1 mM), l-glutamine (2 mM), and glucose (10 mM). Three baseline OCR measurements were taken, followed by three measurements after each injection of oligomycin (1 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 0.5 μM), and rotenone and antimycin A (0.5 mM) (Agilent, 103015-100), respectively. For glycolysis experiments, medium was supplemented with l-glutamine (1 mM). Three baseline measurements were taken for ECAR, followed by three measurements after each injection of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM) (Agilent, 103020-100), respectively. For all analysis, values were normalized to protein concentration before baseline measurements were subtracted. The fold change was calculated relative to DMSO.