Extended data Figure 1: ASS1 deficiency correlates with aspartate utilization by CAD in cancerous and non-cancerous cells.

(A) Schematic flux tracing of the Alpha labeled nitrogen of glutamine (¹⁵N-α-Glutamine) to nucleic acid synthesis via aspartate. (B) The ratio between M+1 labeled/ total level of uracil in fibroblasts is similar between citrullinemia patients and control, n≥3. Error bars represent SER. (C) Labeled levels of M+1 aspartate (left) and M+1 uracil (Right) synthesized from ¹⁵N-α-labeled glutamine, are higher in fibroblasts from CTLN I as compared to fibroblasts from controls and CTLNII patients, n≥3. (D) TCGA analysis of tumor-normal paired tissues for gene expression comparison shows the expression levels of ASL and ASS1 in different cancers. (E) A graph plot generated from the modelling data for the production capacity of metabolites following ASS1 inactivation in each of the NCI-60 cell lines as well as in the generic model. The reddish bars represent the ranking of nucleic acids while the blueish bars represent the ranking of all other metabolites. (F) Correlation analysis of NCI-60 cell lines shows a significant inverse correlation between ASS1 and CAD expression levels. (G) Osteosarcoma (Upper panel) and melanoma (Lower panel) microarray data was obtained from the NCBI EO database (accessions GSE33383 and GSE46517, respectively). Raw expression levels were plotted and significance was computed using t-test on log2-transformed expression levels. The number of patients for each subtype is shown in parenthesis on the left. (H) A western blot for CAD and ASS1 shows higher expression level of CAD in MNNG/HOS human osteosarcoma cell line which has low expression level of ASS1 in comparison to U2OS which has higher expression levels of ASS1. p97 is shown as loading control.