(A) PCK1 expression was measured by qRT-PCR in control shRNA or PCK1 shRNA expressing LS174T cells transduced with empty expression vector or PCK1 expressing vector. Hypoxia cell viability assay with 2 × 105 control shRNA or PCK1 shRNA expressing LS174T cells expressing either empty vector or PCK1 expression vector were cultured under normoxia for 24 hr and then were moved to hypoxic chamber (0.5% O2) for 5 days. PCK1-depleted cells that were generated to re-express PCK1 (rescued expression) exhibited significantly more cell growth than PCK1 depleted cells (p<0.0001, Student’s t-test) (B) Volcano plot showing the metabolite profile of LS174T cells expressing shCTRL or shPCK1 under normoxia. Log2 fold change versus -log10 (p-value) was plotted. Dotted lines along x-axis represent ±log2 (National Cancer Institute, 2019) fold change and dotted lines along y-axis represent -log10 (0.05). All metabolites either significantly enriched or depleted in shPCK1 cells compared to shCTRL are denoted as red points. All other metabolites detected are depicted as gray points. (C) Pyrimidine metabolite levels in LS174T shCTRL versus shPCK1 cells under normoxia (q = 0.79, 0.20, 0.20, and 0.20 in Uridine, UMP, CMP, and CDP, respectively, Student’s t-test, FDR adjusted at Q value of 0.01). Purine metabolite levels in LS174T shCTRL versus shPCK1 cells under normoxia (q = 0.44, 0.36, and 0.36 in Guanosine, IMP, and AMP respectively, Student’s t-test, FDR adjusted at Q value of 0.01). (D) Schematic of U-13C-glutamine stable isotope labeling of metabolites undergoing oxidative metabolism. (E) m+four or m+five fraction labeled metabolites in shCTRL or shPCK1 LS174T cells from 0 to 6 hr of U-13C-glutamine labeling under 0.5% O2. m+four malate, aspartate, and orotate were significantly increased in shPCK1 cells (p=0.003, 0.01,<0.0001 respectively, Student’s t-test, Bonferroni adjusted). (F) Hypoxia cell viability assay in 6 mM Glucose + pyruvate, or aspartate rescue. 2 × 105 LS174T cells expressing either control hairpin or shPCK1 hairpin were cultured under normoxia for 24 hr and then were moved to hypoxic chamber (0.5% O2) for 5 days. Upon exposure to hypoxia, pyruvate(1 mM), aspartate(10 mM) or both pyruvate(1 mM) and aspartate(10 mM) were added to the corresponding cells (p=0.046, 0.002, and 0.0002 in shPCK1 vs shPCK1 plus pyruvate, aspartate, and both pyruvate and aspartate respectively, Student’s t-test, Bonferroni adjusted).