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. 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. Person’s R value was evaluated using ImageJ coloc2 plugin on 25 ROI in three biologically independent experiments (75 ROI in total per condition). I Quantification of the colocalization between LAMP2 and mTORC1 as shown in ( H). Cells were stained against LAMP2 (lysosomal marker, red), mTORC1 (green) and DAPI (blue). H Immunofluorescence microscopy captions 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. 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. E Basal respiration used to drive ATP production as determined by OCR quantification of data obtained in ( D).
Data are mean ± SEM of three biologically independent experiments performed with five replicates. OCR was measured either in basal conditions or after the injection of oligomycin, FCCP, and rotenone/antimycin A. 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. C ATP/ADP ratio of amino acid-starved U2OS cells incubated in the presence or absence of LQ during the indicated times. B ATP/ADP ratio of U2OS cells incubated in absence of amino acid for the indicated time. ATP/ADP ratio of U2OS cells incubated in the presence or the absence of all amino acids for 2 or 72 h. 13 INSERM U1218, Institut Bergonié, Bordeaux, France. 12 Institut Européen de Chimie et Biologie, INSERM U1218, Université de Bordeaux, Pessac, France. 11 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Universidad Pablo de Olavide, Seville, Spain.10 CRUK Beatson Institute, Glasgow, UK.9 Departamento de Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Seville, Spain.8 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Seville, Spain.7 INSERM U1218, Institut Bergonié, Bordeaux, France.6 Service Commun des Animaleries, Animalerie A2, University of Bordeaux, Bordeaux, France.5 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.4 Medical Research Council Cancer Unit, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK.3 Bordeaux Research in Translational Oncology, INSERM U1053, Université de Bordeaux, Bordeaux cedex, France.2 Institut Européen de Chimie et Biologie, INSERM U1218, Université de Bordeaux, Pessac, France.1 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Universidad Pablo de Olavide, Seville, Spain.
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