treated groups. Interestingly, all discriminating metabolites in A549 cells can be observed in PC-9 cells. While the remaining 22 metabolites only exhibited specific changes in PC-9 cells. Among these metabolites, glutamine and glutamate are directly involved in glutamine metabolism. Aspartate14andN-a...
Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine ...
After the treatment, cells were transferred to 96-well plate (4 × 104 cells/well) and the activity of caspases 3/7 was measured using Caspase-Glo® 3/7 bioluminescent assay (Promega), according to the manufacturer's instructions. For the luminescence measurement, microplate reader Synergy II...
Alanine assay kit BioVision Cat#K652 Glutamate assay kit BioVision Cat#K629 Q5® Site-Directed Mutagenesis kit NEB Cat#E0554S Lysosome Enrichment kit Pierce Cat#89839 NE-PER kit Pierce Cat#78833 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat#G7570 CellTox™ Green Cytotoxicity...
2.5. Glutamate and Glutamine Dosages Glutamate and glutamine concentrations were determined by modified GLN1 Kit (Sigma, Saint-Quentin-Fallavier, France). Glutamate concentration was directly evaluated in the sample by dehydrogenation of the L-glutamate to α-ketoglutarate accompanied by reduction of NA...
3.2. Intracellular Glutamine and Glutamate Increase in the Presence of HPV16 E6 Oncoprotein To characterize the glutaminolysis profile of C-33A cells expressing EV, E6, or E7, intracellular glutamine and glutamate metabolites were measured through HPLC assay. Cells were fasted for two hours in medium...
The amount of glutamate and ammonia produced by cells following metformin treatment was evaluated respectively with Glutamate Assay Kit (MAK004; Sigma-Aldrich) and Ammonia Assay Kit (AA0100; Sigma-Aldrich) as previously reported [14]. Absorbance was read through GloMax®-Multi Detection System (...