Dimethyl l glutamate

MDA-MB231 human mammary breast adenocarcinoma (ATCC), HeLa human cervix cancer adenocarcinoma (ATCC) and SiHa human cervix squamous cell carcinoma (ATCC) cells were routinely cultured in DMEM containing 4.5 g/l of glucose and 2 mM of Glutamax, and supplemented with 10% FBS. Assay medium was DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate (D5030, Sigma-Aldrich), reconstituted with 10 mM glucose, 10% FBS, and buffered at pH 7.4 with 3.7 g/L NaHCO₃. Cells were grown at 37°C in a humidified 5% CO₂ atmosphere. Where indicated, cells were treated with 2 mM of L-glutamine (Invitrogen), 2 mM of dimethyl-L-glutamate (Sigma), 2 mM of dimethyl-2-oxoglutarate (Sigma), 2 mM of D-glutamine (Sigma), 5 mM of reduced glutathione (Merck Millipore),10 nM of rapamycin (Sigma), 15 nM of AZD8055 (Selleckchem), 10-100 ng/ml of recombinant human EGF (PeproTech), 20 ng/ml of recombinant IL-6 (Sigma), 1-10µM of Stattic (Santa Cruz), 10 µM of BPTES or 0.25-1 IU/ml of asparaginase (Sigma). Reagents were dissolved either in DMSO or directly in DMEM according to manufacturer’s indications. When DMSO was used, an equal quantity was also added to control medium. All drugs and reagents were administrated to adherent cells in fresh assay medium.