Extended Data Fig. 1: ASS1 expression supports cancer survival under glucose deprivation.
a, The 10 cancer types with high ASS1 (grey, n = 5672; Fig. 1a left panel) demonstrate a significantly lower UCD-score as compared to 10 cancer types with low ASS1 expression (white, n = 6666) following Fig. 1a; (one-sided Wilcoxon ranksum P < 2.2E-16). The box is drawn from top quartile (75-percentile) to bottom quartile (25-percentile) of the data with a horizontal line drawn in the middle to denote the median value. The lowest point of the bottom whisker is the minimum of the data and the highest point of the top whisker is the maximum of the data. Right panel- The figure shows the Spearman rank correlation coefficients (Y-axis) between UCD-score and the expression of each of UC enzymes across TCGA samples. b, Western blot (left) and band intensity quantification relative to GAPDH (right) showing elevation in ASS1, PC and PCK in A549 grown in glucose deficient medium for 48 h. c, Top panel - 4T1 and SW620 cells were grown either in normal medium or in medium without glucose for 48 h. ASS1 levels were analyzed by western blotting. Bottom panel - A western blot showing ASS1 levels in MNNG human osteosarcoma cancer cells grown either in normal medium (control), without serum (NS) or without glucose (NG). d, ASS1 supports glucose-independent survival in murine breast cancer cells and in human colon cancer cells. 4T1 and SW620 cells (expressing either ASS1-shRNA or control GFP-shRNA) were grown either with or without glucose for 48 h (4T1 cells) or 96 h (SW620 cells). Survival was measured by crystal violet staining. Results were normalized to ASS1 expression levels (left panels) or to survival ratio of control cells in glucose-free medium compared to glucose-full medium. n = 3 biological replicates in each group in 4T1 gene expression; n = 3–4 biological replicates in each group in 4T1 survival assay as depicted in the graph; n = 2 biological replicates in each group in SW620 gene expression. In SW620 survival assay presented are technical replicates of a single representative experiment – mean shASS1#1 survival is 30% of control and for shASS1#2 is 74% of control. p value was calculated using two-tailed student’s t-test. In 4T1 gene expression p = 8.8E-07 for control vs. shASS1#1; p = 1.8E-06 for control vs. shASS1#2. In 4T1 survival assay p = 0.032 for control vs.shASS1#1; p = 0.048 for control vs. shASS1#2. In SW620 gene expression p = 0.007 for control vs. shASS1#1; p = 0.006 for control vs. shASS1#2. e, Left panel- RT PCR for ASS1 mRNA levels in A549 control cells versus shASS and shASS1 with ASS1 over expression (OE). Right panel- FACS annexin –shASS1 increases apoptosis. A549 cells were grown in glucose and serum free medium for 72 hrs. Annexin V/7ADD assay was used to determine the percentage of apoptotic and dead cells (% Cell death) in two different clones of shASS1 as compared to control. n = 3–4 biological replicates in each group as depicted in the graph. p value was calculated using Tukey multiple comparisons of means. p = 0.016 for control vs. shASS1#1; p = 0.028 for control vs. shASS1#2. f, Left panel- ASS1 is not required for proliferation of glucose-proficient lung cancer cells. A549 cells (expressing either ASS1-shRNA or control GFP-shRNA) were grown for 72 h. Survival was measured by crystal violet staining. Right panel- A549 cells with shASS1 and ASS1 OE were grown in nutrient deficient medium with and without MDLA. Survival was measured using quantified crystal violet staining. n = 3 biological replicates in each group. p value was calculated using two-tailed student’s t-test. p = 0.038. g, Bar plot showing the association between ASS1 expression and patient survival. Hazard ratio is shown in log10 scale (Y-axis) for 25 cancer types (those cancer types with statistical significance based on logrank test is marked black). The number of samples for each cancer type are listed in the Source Data.
