Figure 1

Metabolic changes in three NSCLC cell lines upon TGFβ-induced EMT. A549, HCC827, and NCI-H358 lung cancer cells were cultured in the presence of 2 ng/ml TGFβ for 2 to 5 weeks to induce EMT. The following aspects of both the parental (P) and mesenchymal (M) cells were characterized. (A) Morphological changes of cells. (B) The expression of the epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin, Zeb1, and Snail). (C) Erlotinib sensitivity of HCC827 and GDC-0973 sensitivity of A549 parental and mesenchymal cells. The cells were treated with the EGFR kinase inhibitor erlotinib or MEK inhibitor GDC-0973 for 3 days, and viability was measured using a CellTiter-Glo assay. (D) Glycolysis/OXPHOS ratio, defined by PPR/OCR and measured using the Seahorse metabolic analyzer. Average results from three to four independent experiments are shown. (E, F) Cellular glutamine (E) and glutamate (F) concentrations as measured by mass spectrometry. Each data point is from five separate biological samples generated at the same time. The boxes represent 10–90 percentile. (G) Glutamate secretion per cell during 24 h. Average of data from six wells in one experiment, which is representative of three independent experiments, is shown. (H, I) Cells were incubated with growth media containing ¹³C-U-glucose overnight, and then subjected to LC-MS analysis. (H) Glucose to glutamate contribution was plotted based on the percentage of (M + 2) glutamate in the total glutamate pool. (I) Glucose to TCA cycle contribution was plotted based on the percentages of (M + 2) citrate, (M + 2) α-ketoglutarate and (M + 2) malate in each individual metabolite’s total pool. For all panels, data are plotted as mean ± SEM. *p < 0.05; **p < 0.01, unless otherwise specified.