Fig. 3

Crotonate treatment leads to reduced glycolysis and enhanced TCA cycle. A Key metabolic intermediates in glycolysis and the TCA cycle are shown. Red, intermediates measured by HPIC-MRM-MS/MS. B Extracellular acidification rate (ECAR) analysis of untreated H9-LTR7 GFP cells, endodermal cells, and reporter hESCs treated with 10 mM crotonate for 12 days. Glucose (10 mM), oligomycin (1 mM), and 2-deoxyglucose (2-DG, 50 mM) were added as indicated. C Glycolytic rates and glycolytic capacity (ECAR following oligomycin) for untreated H9-LTR7 GFP cells, endodermal cells, and reporter hESCs treated with 10 mM crotonate for 12 days. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. D, E Reporter hESCs were cultured in crotonate-containing media (10 mM) for 12 days before targeted metabolomic analysis. Untreated cells and endodermal cells differentiated from the reporter hESCs were used as controls. Levels of the measured intermediates were normalized to standards. The results for several glycolytic intermediates and the end product pyruvate were compared across samples in a heatmap (D) or individually plotted (E). Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05; **p < 0.01. F, G Based on metabolomic data, results for several TCA cycle intermediates were compared across samples in a heatmap (F) or individually plotted (G). Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05; **p < 0.01