Extended Data Fig. 2: ASS1 promotes nutrient-free cancer cell survival through pyruvate utilization for serine and glycine synthesis.
a, ASS1 is required for pyruvate utilization for gluconeogenesis in glucose-starved lung cancer cells. A549 cells (expressing either ASS1-shRNA or control GFP-shRNA) were grown in a glucose and serum-free medium with or without the addition of 1 mM pyruvate for 72 h. Survival was measured using crystal violet staining. n = 3 biological replicates in each group. p value was calculated using matched one-sided Wilcoxon ranksum test. P = 0.05 in control. b, Pyruvate accumulates in ASS1-deficient cancer cells under low-nutrient conditions. A549 cells (expressing either ASS1-shRNA or control GFP-shRNA) were grown in the presence or absence of glucose and serum for 24 h and quantified by liquid chromatography–mass spectrometry (LC-MS/MS) metabolomic profiling. n = 6 biological replicates in each group. p value was calculated using two-tailed student’s t-test. p = 0.013 for the difference between the groups under no glucose state. c, Glycine is produced in nutrient-starved lung cancer cells when glucose is depleted. A549 cells were grown with or without glucose and serum (Low Nutrient) for 48 h. Cells were incubated with [U-13C] glutamine for the last 6 h of culture. Shown for glycine are the total levels quantified by GC/MS as the sum of areas under the curve (AUC) measurement for the different masses/ M + 0; shown for glucose are total levels as AUC. n = 3 biological replicates in each group. p value was calculated using two-tailed student’s t-test. p = 3.1E-05 for glycine labelling and 1.3E-04 for total glucose levels under low nutrient conditions compared to regular medium. d, ASS1 supports serine and glycine production from glutamine under nutrient deprivation. A549 cells were grown for 48 h followed by incubation with [U-13C] glutamine for 6 h. Shown is the isoropologue distribution of [U-13C]-labelled serine and glycine as quantified by GC/MS under low nutrients vs. normal medium in control vs. shASS1. n = 4 biological replicates in each group. p value was calculated using two-tailed student’s t-test. For serine: p = 0.0003 in M + 2 and 0.018 in M + 3; For glycine: p = 0.014 for M + 1. e, ASS1 inhibition does not alter total serine (left panel) or glycine levels (right panel). For the total levels of serine and glycine, A549 cells were grown for 48 h with or without glucose and serum followed by culture with [U-13C] glutamine for the last 6 h of incubation. Shown are total levels quantified in GC/MS by area under the curve (AUC) measurement. n = 4 biological replicates in each group. p value was calculated using two-tailed student’s t-test. f, ASS1 supports the synthesis of gluconeogenesis intermediates and glycine production under glucose deprivation.4T1 cells were grown with no glucose for 48 h. Metabolites were analysed by LC-MS. PEP was measured by AUC normalized to protein concentration. Shown is the increment in PEP levels in glucose-deprived cells relative to cells grown in normal conditions (Left panel), and the levels of [U-13C]-labelled mass pools/M + 0 of 3PG and glycine, as quantified by LC/MS (Right panel). n = 3–5 biological replicates in each group as depicted in the graphs. p value was calculated using two-tailed student’s t-test. For PEP p = 0.04; for 3PG p = 0.017 in no glucose; for glycine p = 0.005.
