Figure 2. ASS1-deficient tumors have increased proliferation rate and an increase in pyrimidine levels.

(A) Upper panel- ASS1 is ranked within the top 24 genes out of 14,000, that show a significant inverse correlation with proliferation rate among the 16 NCI-60 cancer cell lines in which ASS1 is downregulated; Spearman rank correlation between the expression of each gene and its associated proliferation rates. Lower panel- A magnifying view of the correlation between proliferation rate and ASS1 expression levels. (B) Predicted differences in the production rate of biomass components following the inactivation of ASS1. The production of nucleic acids marked in arrows, is predicted to have a large increase following ASS1 inactivation in the majority of the NCI-60 cell-lines. The figure represents the results obtained using the LOX IMVI cell-line model, however the same results were seen for all NCI- 60 cell lines as well as in using the human generic model. The models used are based on a series of simplifying assumptions as described by us previously in details¹⁰ (C) Analysis of the TCGA database of matched tumor-normal tissue pairs showing that CAD expression is elevated significantly in tumors with ASS1 downregulation as compared to normal tissue, (p-Value<0.05). (D) An immunoblot of osteosarcoma cell lines showing decreased expression of ASS1 compared to the loading control GAPDH in MNNG/HOS as compared to U2OS. (E) Osteosarcoma cells with ASS1 downregulation have a significant increase in pyrimidine levels as measured by LC/MS, n≥3 (*p-Value<0.05; ***p-Value<0.0005). Error bars represent SER (F) Osteosarcoma cells with ASS1 downregulation have a significant increase in uracil, n=3 (*p-Value<0.05). Error bars represent SER (G) Osteosarcoma cells with ASS1 downregulation have a significant increase in proliferation as measured by MTT assay, (***p-Value<0.0005).