Fig. 3From: ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolismCombined expression of ASS1 and ASL in ccRCC cells suppresses growth and alters the metabolic landscape in a catalytically dependent manner. A Combined ectopic expression of ASS1 and ASL in 786-O polyclonal population. HSP90 serves as the loading control, while HK-2 cells and the normal kidney are normal controls. B ASS1+ ASL expression suppresses growth in 786-O cells in a 2D growth assay. Error bars represent SEM of 7 wells, and cells were grown in 1% FBS in complete DMEM. ***p < 0.001. C 3D soft agar colony-forming assay with 786-O cells expressing empty vector control and ASS1+ASL cDNA. Error bars represent SEM of 3 wells. *p < 0.05. D Steady-state amino acid levels in 786-O cells expressing empty vector and ASS1+ASL cDNA. Error bars represent SEM of 3 technical replicates. **p < 0.01. E Steady-state aspartate and glutamate levels in 786-O cells expressing empty vector and ASS1+ASL cDNA. Error bars represent SEM of 3 technical replicates. **p < 0.01. F Schematic representation of 15N2-glutamine labeling into aspartate, orotate, and argininosuccinate. Argininosuccinate synthase (ASS1), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD enzyme complex). The filled-in squares represent heavy nitrogen (15N) while clear circles represent carbon atoms (12C). G Schematic representation of 13C6-glucose labeling into aspartate. The filled-in circles represent heavy carbon atoms (13C). H EdU incorporation assay with 786-O cells expressing empty vector and ASS1+ASL cDNA. Error bars represent SEM of 3 technical replicates. *p < 0.05. I Combined ectopic expression of wild-type and catalytically dead mutant ASS1 and ASL in 786-O polyclonal population with mutant sequence. J ASS1+ASL-mediated growth suppression in 786-O cells is dependent on their catalytic activity as seen in a 2D growth assay. Error bars represent SEM of 6 technical replicates. ***p < 0.001. K 786-O cells expressing ASS1+ ASL cDNA were cultured with exogenously provided pyrimidines (cytidine, thymidine, and uridine) at indicated concentrations. 786-O cells expressing empty vector are shown as control. Error bars represent SEM of 6 technical replicates. **p < 0.01. L 786-O cells expressing ASS1+ASL cDNA were cultured with 150 μM exogenously provided aspartic acid. 786-O cells expressing empty vector are shown as control. Error bars represent SEM of 6 technical replicates. ***p < 0.001Back to article page