Extended data figure 4: Cancers with ASS1 downregulation are addicted to aspartate.

(A-B) Analysis of the TCGA data base of matched tumor-normal pairs showing no significant difference in the expression level of citrin in tissues with a high base line expression of citrin, (A) and significant elevation in tumors in which the normal tissue has low basal expression of citrin (B) (*p<0.001). (C) Immunoblot showing the expression level of citrin in osteosarcoma cells following si-citrin.(D) Labeled and unlabeled aspartate (Upper panel) and uracil (Lower panel) are elevated significantly in cancers with ASS1 downregulation and are comparable to control in cells with both ASS1 and Citrin downregulation; n≥3. Error bars represent SER. (E) Kaplan-Meier (KM) survival analysis for 2 different cancer types; BRCA- breast cancer (Upper panel) and LUSC- lung squamous cell carcinoma (Lower panel), showing poor survival trend for cancers with low ASS1 and high citrin. For each cancer type the KM-plot indicates the survival rates of 4 patients group: ASS1 low expression - citrin high expression; ASS1 low expression; citrin high expression; none of the above groups. Analysis was performed for the cancer types for which there was sufficient survival data. (F) A quantification graph of a western blot showing decreased CAD and S6K phosphorylation following treatment of U2OS with si-citrine. Error bars represent SER. (G) Proximity ligation assay showing increased proximity between CAD and citrin following ASS1 knockdown in U2OS cells (Upper panel-red dots). The left and middle pictures show the proximity between ASS1 and CAD to citrin in U2OS infected with empty vector while the right picture shows the proximity between CAD and citrin following infection of U2OS with shASS1. The Lower panel shows quantification of proximity ligation assays performed on U2OS infected with either empty vector (EV) or with shASS1 using antibodies for citrin, ASS1 and CAD. The pictures were quantified using ImageJ.