Fig. 3.

Knockdown of pyruvate carboxylase (PC) expression does not alter the levels of glycolytic intermediates glucose-6-phosphate plus fructose-6-phosphate or fructose-biphosphate from U-¹³C₆-glucose or U-¹³C₅-glutamine in MDA-MB-231-derived cell lines. PC 847 2C and PC 2096 4B or a scramble shRNA control cell line were maintained in DMEM cell culture medium (contains 25 mM glucose and 4 mM glutamine) and 10% FBS for four or more days. Cells were then maintained in DMEM medium containing either 10 mM [U-¹³C₆] glucose or 2 mM [U-¹³C₅]glutamine for 1 h before metabolism was stopped and cells were analyzed by LC-MS/MS as described under Experimental Procedures. A, The levels of glucose-6-phosphate (G6P) plus fructose-6-phosphate (F6P) before the uniformly labeled glucose or glutamine was added (zero time point). B, The level of fructose-1,6-bisphosphate (FBP) before the uniformly labeled glucose or glutamine was added (zero time point). C and D, Upon adding the labeled glucose or glutamine to the culture media, the metabolites were extracted and analyzed by LC-MS/MS spectroscopy. Fractions of different isotopomers of U-¹³C glucose-6-phosphate plus U-¹³C fructose-6-phosphate or fructose-biphosphate are shown within the bars. AUC indicates “area under the curve”.