Extended Data Fig. 3: ASS1 promotes gluconeogenesis via nitrosylation.
a, NO donors (SNAP), rescue survival of starved ASS1-inhibited lung cancer cells. A549 cells were grown for 72 h with and without 10 mM SNAP. Survival of shASS1 cells relative to control cells was determined using crystal violet staining. n = 3 biological replicates in each group. p value was calculated using two-tailed student’s t-test. p = 0.047. b, NO is required for proliferation of ASS1-overexpressing cancer cells. 4T1 cells were grown for 48 h with or without the addition of 10mM L-NAME. Cell viability was measured using crystal violet staining. n = 3 biological replicates in each group. p value was calculated using two-tailed student’s t-test. p = 0.015. c, Quantification of the westrn shown in Fig. 3g demonstrating ASS1 necessity for starvation-induced S-nitrosylation of gluconeogenesis enzymes in nutrient-deficient lung cancer cells. A549 cells (expressing either ASS1-shRNA or control GFP-shRNA) were grown in the absence of glucose and serum for 48 h. Detection of protein nitrosylation was performed using the SNO-RAC method followed by a western blot. n = 3 biological replicates in each group. p value was calculated using two-tailed student’s t-test. p = 0.036 for PC and 0.002 for PCK2. d, S-nitrosylation of gluconeogenic enzymes in nutrient-proficient lung cancer cells. A549 cells were grown in the presence of glucose and serum for 48 h. Detection of protein nitrosylation was performed using the SNO-RAC method followed by a western blot and quantification. e, ASS1 is required for starvation-induced S-nitrosylation of gluconeogenic enzymes in livers of fasting mice. ASS1f/f Alb-CRE and control mice were fed either regularly or fasted for 16 h. Detection of protein nitrosylation in the liver was performed using the SNO-RAC method followed by a western blot and quantification. n = 3 mice in each group. p value was calculated using two-tailed student’s t-test. p = 0.042.
