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Glycolytic rate was determined by measuring the conversion of [5-3H]glucose (GE Healthcare, Piscataway, N.J.) to 3H2O according to a published procedure (Ashcroft et al., Biochem J. 126, p. 525-532 (1972)). Briefly, LNCaP cells were seeded in six-well plates (4×105 cells/well) and then treated 24 h later with 10 mM 2-DG or 10 μM OSU-CG12 for various intervals. After washing with PBS, cells were trypsinized and resuspended in 500 μL of Krebs buffer [25 mM NaHCO3, 115 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 0.25% BSA (pH, 7.4)] containing 1 mM nonradioactive glucose and 5 μCi/mL [5-3H]glucose for 1 h at 37° C. Aliquots from each treatment group were added to 0.2 N HCl in open tubes that were placed upright in scintillation vials containing 1 mL of H2O. The vials were sealed and H2O produced by glucose consumption was equilibrated with H2O outside the tube for a minimum of 24 h at room temperature. The amount of 3H retained in the tube and the amount that had diffused into the surrounding H2O by evaporation and condensation were determined separately by using a scintillation counter LS6500 (Beckman). [5-3H]glucose-only and 3H2O-only standards were included in each experiment for calculation of the rate of conversion of [5-3H]glucose to H2O using the following equation: glucose utilized (pmol)=[3H] water formed (d.p.m)/[5-3H]glucose (d.p.m/pmol)].

1. washAfter washing with PBS, cells
2. additioncontaining 1 mM nonradioactive glucose and 5 μCi/mL [5-3H]glucose for 1 h at 37° C
3. additionAliquots from each treatment group were added to 0.2 N HCl in open tubes that
4. additioncontaining 1 mL of H2O
5. customThe vials were sealed