of spot s in a gel containing n spots. Raw spot values were normalized using the Cisplatin mechanism softwares Inhibitors,Modulators,Libraries ratio option according to the following equation, Protein identification using database searching Proteins were identified by searching the databases of the NCBInr and SwissProt using ProteinPilot 2. 0 with paragorn the mean of spot s. Changes in average volume larger than 40% of the average spot volume and the significance level of P 0. 05 was the criterion used for excision. Four replicates were used for each control, lovastatin lactone or acid treatment, respectively. In gel digestion Proteins from excited gels spots were digested using a modification of Inhibitors,Modulators,Libraries the method by Havlis. Briefly, spots algorithm software.

Parameters used in the database search were as follows, biological modifications, fixed modification, iodacetamide alkylation of Cys, detected protein thresh old, more than 1, thorough ID. Cell extraction for nuclear magnetic resonance spectroscopy For NMR experiments, the cells were incubated with 5 mmol L glucose for the last five hours Inhibitors,Modulators,Libraries prior to the perchloric acid extraction. All cell extractions were performed using a previously published PCA extraction protocol that allowed for separation of water soluble and lipid fractions. Lyophilized water soluble cell extracts were re dissolved in 0. 5 mL of deuterium oxide, centri fuged and the supernatants neutralized to pH 7. 2 in order to allow for precise chemical shift assignments. Lipid frac tions were re dissolved in a 1 mL CD3OD CDCl3 mixture.

NMR spectroscopy High resolution 1H and 13C NMR experiments were performed using a Varian INOVA NMR 500 MHz spec trometer equipped with a 5 mm HCN PFG probe. For 1H NMR analysis of water soluble extracts we have used fully relaxed spectra with a standard water presaturation Inhibitors,Modulators,Libraries pulse program, Inhibitors,Modulators,Libraries whereas for analysis of lipids no presaturation pulse was used. Spectra were obtained at 12 ppm spectral width, 32 K data arrays, and 64 scans with 90 degree pulses applied every 14. 8 seconds. The pool size of metabolites was determined based on fully relaxed 1H NMR spectra of extracts using trimethylsilyl propionic 2,2,3,3, d4 acid as an external standard and chemical shift reference.

The absolute con centrations of each metabolite were deter especially mined and normalized according to cell wet weight, as previously described and calculated using the following equation, where integralmet is integral of respective metabolite signal divided by the number of protons, integralTSP is integral of TSP signal divided by the number of protons, is TSP nominal concentration, VS is sample volume, wet weight is sample weight. 13 C NMR spectra with proton decoupling were recorded using the C3 lac tate peak at 21 ppm as chemical shift reference. For quantifi cation of absolute concentrations of 13C metabolites, calculations were made according to.