Figure 3

(a) Hyperosmotic stress inactivates mTORC1 in a dose-dependent manner. MEFs were exposed to normal or hyperosmotic culture media for 15 min. Osmolality of the medium was elevated by increasing NaCl concentration by 33–200 mM above baseline. (b) Energetic stress inactivates mTORC1 in a dose-dependent manner. MEFs were treated with the indicated amounts of 2-DG for 30 min. (c) Hypoxic stress inactivates mTORC1 in a dose-dependent manner. MEFs were treated for 24 h with the indicated amounts of CoCl₂, a compound which partially mimics hypoxia. (d) mTORC1 is inactivated by changes in the pH of the culture media. MEFs were treated with pH-adjusted media for 30 min. (e,f) TSC2 relocalizes to lysosomes upon exposure of cells to all the stresses tested singly. MEFs were cultured in full medium (+aa +serum). Cells were exposed to each stress as indicated in the figure and as described in a–d, and TSC2 localization was analysed by immunostaining. LAMP2 staining was used as a lysosomal marker. Representative magnified insets are shown on the right (top: TSC2; middle: LAMP2; bottom: merged). The degree of co-localization between TSC2 and LAMP2 (automatically thresholded MCC) is shown in f as a mean±s.e.m. ***P<0.001 for comparison to control (Ctrl), using one-way ANOVA. Note that each stress stimulus is sufficient to cause lysosomal recruitment of TSC2 in the presence of serum and amino acids. For all treatments, data representative of at least three independent biological replicates are shown. See also Supplementary Figs 6–10.