Fig. 4: Gαq/11 is required to reactivate the mTORC1 pathway and thus inactivate autophagy in response to nutrient recovery.

a WT and Gαq/11 KO MEFs were either maintained in the presence of 10% serum (control condition) or subjected to 24 h of starvation (0.1% FBS medium), followed by stimulation for 18 h with increasing doses of serum. WT vs. Gαq/11 KO MEFs P value (0% FBS) = 0.0049; P value (0.1% FBS) = 0.0099; P value (1% FBS) = 0.0330. b WT and Gαq/11 KO MEFs were either maintained in the presence of 10% serum or subjected to a 30 min treatment with EBSS, followed by stimulation with a full amino acid mix for the indicated times. WT vs. Gαq/11 KO MEFs P value (0 min) = 0.0112; P value (15 min) = 0.0377; P value (30 min) = 0.0090; P value (60 min) = 0.0177. Autophagy markers (LC3-I/II) were analyzed by western blot (see quantifications in Supplementary Fig. 8a and c) and the activation of mTORC1 and AMPK pathways by assessing the phosphorylation status of downstream targets of mTORC1 (S6 ribosomal protein, see Supplementary Fig. 8b and d for additional readouts of this cascade) or AMPK, respectively. Tubulin was used as a loading control. Phospho-S6 data (mean ± SEM of three independent experiments) were normalized using total S6 ribosomal protein. Representative blots are shown. a, b Statistical significance was analyzed using two-sided unpaired t-test. For all P values, *P < 0.05, **P < 0.01. c Altered intracellular positioning of lysosomes in Gαq/11 KO cells. Representative confocal micrographs of LAMP1 (red) and DAPI (blue) in WT, Gαq/11 KO and Gαq/11 KO + Gq MEFs in control conditions, after 30 min in amino acid-free medium (EBSS) and upon re-exposure to amino acids for 15 min (EBSS plus AA). Images are maximum intensity projections acquired using Zeiss LSM780 confocal microscope using a ×63 Oil (NA = 1.4) oil immersion lends. Scale bar, 10 µm. Image on the right shows the quantification analysis of lysosome distribution. The fluorescence intensity density per ring was measured and normalized as detailed in “Methods” and Supplementary Fig. 9. Control (Wt, n = 30; Gαq/11 KO, n = 30 and Gαq/11 KO + Gq, n = 20); EBSS (Wt, n = 30; Gαq/11 KO, n = 30 and Gαq/11 KO + Gq, n = 20); EBSS + aa (Wt, n = 30; Gαq/11 KO, n = 28 and Gαq/11 KO + Gq, n = 22). Data are mean ± SEM from the indicated cells for each condition from three independent different experiments. Statistical significance was analyzed using one-way ANOVA Sidak’s multiple comparisons test. For all P values, ****P < 0.0001. d–f The specific Gαq/11 inhibitor YM-254890 impairs reactivation of the mTORC1 pathway. d WT MEFs pretreated with YM-254890 (10 µM, 1 h) were maintained in the presence of 10% serum (control condition) or subjected to a 10 min treatment with EBSS, followed by stimulation with a full amino acid mix for 30 min and analysis of mTORC1 cascade readouts. Representative blots of three independent experiments are shown. e Diagram for mTORC1 pathway analysis in liver explants from fed mice subjected to YM-254890 (40 µM) or Rapamycin (500 nM) treatments for 1 h. The images used in this diagram are from Servier Medical Art (http://smart.servier.com/). f Normalized p-S6 data (mean ± SEM of four mice) and a representative blot are shown. Statistical significance was analyzed using two-sided unpaired t-test. For all P values, *P < 0.05. Control vs. YM-254890 (YM), P value = 0.0260; Control vs. Rapamycin (Rapa), P value = 0.322. n.s. nonsignificant. Source data are provided as a Source Data file.