Fig. 5

Metabolic analysis of tumors from mice treated with CB-839 and metformin alone and in combination. a ¹H NMR representative spectra of tumor extracts under the 4 conditions investigated herein, displaying changes in the intensity of glutamate and glutamine resonances attributable to treatment. Intensities have been normalized to the TSP signal and to the tissue weight. Spectra displaying the multiplets arising from the protons linked to the C2 for both Glu and Gln. b Quantification of glutamate and glutamine from ¹H NMR data. c Volcano plot displaying the main metabolites arising from the analysis vehicle vs CB-839+metformin as colored dots matching the pathway they belong to (see Additional file 1: Table S1 for the list of metabolites). d Roadmap of ¹³C-glutamine derived carbons throughout both, oxidative and reductive routes of TCA. e Tumor metabolites were analyzed by LC-MS after tail vein injections of ¹³C-Glutamine. m+0, unlabeled; m+1, m+2, m+3, m+4, m+5, and m+6 represent the degree of m/z increase due to ¹³C labeling. The results are presented as relative contribution of each isotopologue to the total pool at the metabolite. Data are displayed as the mean ± SD (n = 3–5). f 1d-HSQC spectrum displaying the peak intensity of protons attached to ¹³C atoms after ¹³C-glutamine injections revealing the differences arising from each treatment as the intensities of labeled metabolites. The intensities of the resonance peaks displayed were normalized to the TSP signal and to the tissue weight. g The same as D, but after ¹³C-glucose injection. Pyruvate enrichment analyzed by LC-MS and quantification of ¹³C-glucose-derived lactate (h) from NMR analysis. i Growth of MG63.3 cell line under CB-839, rotenone, and combination. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by multiple comparisons “vehicle vs each treatment”)