Extraction of cellular metabolites

Cells were seeded (2 × 10⁶ cells per flask) in 75 cm² culture flasks until 60–70% confluent, then treated with IC₅₀ doses of the respective inhibitors for 24 h. The cells (typically 10⁷ per sample) were then detached by treatment with trypsin and after addition of ice cold media transferred to 1.5 ml microfuge tubes. Briefly, cells were pelleted by centrifuging at 500 g for 5 min at 4 °C and washed twice with ice-cold isotonic saline to remove extracellular phosphate. The metabolites were extracted by a two-solvent system¹⁹. To the pellets, 20 μl of 1-aminopropylphosphoric acid (0.3 μM) (³¹P-NMR standard), 10 μl of 10 mM EDTA (10 mM) and 0.375 ml of methanol/chloroform (2:1) were added and left on the ice for 1 hour after thorough shaking. Thereafter phase separation was accomplished by adding 0.125 ml of chloroform and Tris-HCl buffer (10 mM) solution (pH 7.4). The mixtures were centrifuged at 1000 g for 10 min at 4 °C. ³¹P-NMR spectroscopy was carried out on the aqueous phase after addition of deuterium oxide (final concentration 10%). A protein assay was carried out, for normalising the NMR data, on the precipitated tissue at the interface of the phases after drying and dissolving overnight in 0.5 ml of NaOH (1 M) and neutralizing with HCl.