Fig. 7: Gαq/p62 association is regulated by nutrient availability and mTORC1 and autophagy modulation by Gαq correlate with its ability to interact with p62.

a CHO cells were transfected with HA-p62 and Gαq constructs, starved with 0.1% FBS and treated with a combination of NH₄Cl (20 mM) and leupeptin (100 µM) (NH₄Cl/Leu) (N/L) during the last 4 h of the starvation period, or with EBSS or glucose-free media for the indicated times (b). An LC3-I/II blot is shown to confirm the inhibition of autophagy by N/L. In both (a, b), co-immunoprecipitation assays and expression controls in lysates were performed as described under “Methods”. Representative blots of three independent experiments are shown. c Basal activation status of the mTORC1 cascade and autophagy levels correlates with the ability of Gαq mutants to interact with p62. The basal activation status of the mTORC1 pathway in cells growing under nutrient-sufficiency conditions (10% FBS) was checked by analyzing the phosphorylation status of the S6 ribosomal protein in wild type MEFS, Gαq/11 KO MEFs and MEFs stably expressing in a Gαq/11 KO background either Gαq wt (+Gq MEFs) or a Gαq mutant (+Gq-EEAA) with decreased Gαq/p62 interaction. S6 phosphorylation data from immunoblot analysis (mean ± SEM of 11 (WT), 9 (Gαq/11 KO and Gαq/11 KO + Gq), and 8 (Gαq/11 KO + Gq-EEAA) independent experiments) were normalized using total S6 ribosomal protein and expressed as fold change with respect to the WT MEFs population. Basal autophagy levels were checked by analyzing LC3 protein levels by western blot and LC3-II/LC3-I ratios (mean ± SEM of four independent experiments) expressed as fold change with respect to the WT MEFs population. Statistical significance was analyzed using two-sided unpaired t-test. For all P values, *P < 0.05, ****P < 0.001. WT vs. Gαq11KO + Gq-EEAA, P value = 0.0402. d Endogenous mTOR immunoprecipitation was performed in Gαq/11 KO + Gq and Gαq/11 KO + Gq-EEAA expressing MEFs maintained under nutrient-sufficiency conditions (10% FBS). mTOR immunoprecipitates and total lysates were analyzed by western blot with specific antibodies to determine the components of the complex and overall cell expression levels, respectively. The blots shown are representative of three independent experiments. e mTORC1 reactivation and autophagy modulation in response to amino acid recovery after starvation correlate with the ability of Gαq to interact with p62. As shown in the experimental outline diagram, all cell lines were treated or not with EBBS for 30 min and then stimulated with a full amino acid mix for 15 min. mTORC1/autophagy pathways modulation were analyzed by western blot as in previous figures. S6 phosphorylation data (mean ± SEM of three (control and EBSS + 15 aa) and four (EBSS) independent experiments) were normalized using total S6 ribosomal protein and expressed as fold change over values in Gαq/11 KO MEFs population in each experimental condition. Statistical significance was analyzed using two-way ANOVA with Bonferroni multiple comparison test. Gαq/11 KO (EBSS + 15 aa) vs. Gαq/11KO + Gq (EBSS + 15 aa), P value = 0.0003. Source data are provided as a Source Data file.