Fig. 1: Glutaminolysis sustains the production of ATP to inhibit AMPK and to activate mTORC1.

A ATP/ADP ratio of U2OS cells incubated in the presence or the absence of all amino acids for 2 or 72 h. Fed cells (C) are used as control. B ATP/ADP ratio of U2OS cells incubated in absence of amino acid for the indicated time. C ATP/ADP ratio of amino acid-starved U2OS cells incubated in the presence or absence of LQ during the indicated times. D OCR analysis by Seahorse^® technology of amino acid-starved U2OS cells incubated in the presence (blue) or absence (purple) of LQ during 72 h. OCR was measured either in basal conditions or after the injection of oligomycin, FCCP, and rotenone/antimycin A. Data are mean ± SEM of three biologically independent experiments performed with five replicates. E Basal respiration used to drive ATP production as determined by OCR quantification of data obtained in (D). F Immunoblot of mTORC1 activity markers (S6K, S6, and 4EBP1 phosphorylation) and AMPK phosphorylation of U2OS cells incubated with or without amino acids, in the presence or absence of LQ during 72 h. G Immunoblot of mTORC1 activity markers (S6K and S6 phosphorylation) and AMPK phosphorylation of amino acid-starved U2OS cells incubated in the presence or absence of LQ during 24, 48, or 72 h. H Immunofluorescence microscopy captions of U2OS cells incubated with or without amino acids, in the presence or absence of LQ during 72 h. Cells were stained against LAMP2 (lysosomal marker, red), mTORC1 (green) and DAPI (blue). Scale bar represents 10 µm. I Quantification of the colocalization between LAMP2 and mTORC1 as shown in (H). Person’s R value was evaluated using ImageJ coloc2 plugin on 25 ROI in three biologically independent experiments (75 ROI in total per condition). J Immunoblot of mTORC1 activity markers (S6K and S6 phosphorylation) and AMPK phosphorylation of amino acid-starved U2OS cells incubated in the presence or absence of LQ, with or without AICAR, during 72 h. K Immunoblot analysis of mTORC1 activity marker (S6 phosphorylation) of U2OS cells expressing a myc-tagged, constitutively active AMPK mutant in the presence or absence of amino acids and LQ as indicated. L Immunoblot of autophagy (p62 and LC3-I/II) and mTORC1 (S6K and S6 phosphorylation) markers of amino acid-starved U2OS cells incubated in absence or presence of LQ, AICAR, metformin or A769662 for 72 h, as indicated. M Fluorescence microscopy captions of GFP-LC3 expressing amino acid-starved U2OS cells incubated in the presence or absence of LQ, with or without AICAR, metformin or A769662 for 72 h. Autophagosome formation upon GFP-LC3 aggregation was assayed using confocal microscopy. The scale bar represents 10 µm. N Quantification of the number of GFP-LC3 dots per cell of captions obtained in (M). >100 cells were counted per experiment. O Immunoblot analysis of autophagy markers (p62 and LC3I/II) of U2OS cells treated with LQ, AICAR and/or Bafilomycin A1 as indicated for 72 h. Graphs show mean values ± SEM (n = 3 biologically independent experiments). *p < 0.05 (ANOVA analysis followed by a post hoc Bonferroni test). Source data are provided as a Source Data file.