Fig 2: PI3K/Akt/mTOR/autophagy are insensitive to starvation in senescent cells. (a) Immunoblot analysis of mTORC1 and autophagy activity in control and senescent (Sen(IR); 30 d after 20 Gy x-ray irradiation) primary human fibroblasts after overnight serum starvation with or without 1-h amino acid starvation. (b) As in panel a, but senescence was achieved by transduction of cells with oncogenic mutant B-RAFV600E (oncogene-induced senescence, OIS). (c) Immunoblot analysis of signaling cascades upstream of mTORC1 after starvation protocols as described in panel a. (d and e) Proliferating or replicative senescent (Sen(Rep)) cells were treated as in a, fixed, and immunostained for antibodies against TSC2 and Lamp1. White arrows indicate recruitment of TSC2 to lysosomes (d). Colocalization between Lamp1 and TSC2 (e) was analyzed. Error bars represent SEM; all experiments, n = 3 (for colocalization, at least 10 cells imaged per experimental repeat). Student’s t test performed between groups: *, P < 0.05; ***, P < 0.001; NS, not significant. Bars, 10 µm.

Fig 3: AKT activity in p53zdf1/zdf1 and tsc2;p53 compound mutant tumors. (A–F) Phospho-tuberin (p-Tsc2) staining in p53zdf1/zdf1 and tsc2;p53 compound mutant zebrafish. (B,C and E,F) Higher magnification of A and D, respectively. (C,F) Phospho-tuberin (Ser939, red); (B,E) phospho-tuberin (red) merged with DAPI (blue). (G,J) Total Akt immunofluorescence, (H,K) phospho-Akt (Thr308) immunofluorescence and (I,L) phospho-Akt (Ser473) immunofluorescence in p53zdf1/zdf1 (G–I) and tsc2;p53 compound mutant (J–L) zebrafish. M, muscle; T, tumor. Scale bars: 100 µm.

Fig 4: Hypoxia inhibits mTORC1 and causes lysosomal accumulation of TSC2.(a) Hypoxia inactivates mTORC1. MEFs were incubated in a hypoxic chamber (1% O2) for the indicated times before lysis, and mTORC1 activity was assayed by immunoblot using the indicated antibodies (representative of three independent biological replicates). (b,c) TSC2 relocalizes to lysosomes upon hypoxia. MEFs were incubated in a hypoxic chamber (1% O2) for the indicated times before fixation, and TSC2 localization was assayed by immunofluorescence. Representative magnified insets are shown on the right (top: TSC2; middle: LAMP2; bottom: merged), and quantification in c was performed as in Fig. 3e,f (shown as mean±s.e.m.). ***P<0.001, *P<0.05 for comparison with control (Ctrl) cells, using one-way ANOVA (representative of two independent biological replicates).

Fig 5: PRL-3-driven mTOR activation correlates with increased Akt-TSC2-Rheb signalling.(a) HCT116 cells overexpressing EGFP vector only (Vec), EGFP-tagged wild-type PRL-3 (PRL-3) or EGFP-tagged catalytic-inactive PRL-3 C104S (C104S) were cultured for 24 h in full media under normoxia (Ctrl), hypoxia (Hx), or serum-free (SF) conditions, prior to lysis and western blot analysis with the indicated antibodies. (b) HCT116 cells overexpressing Vec, PRL-3 or C104S were cultured for 1 h in full media under normoxia (Ctrl) or amino-acid starved (AA-) conditions, prior to analysis as in (a). (c) HCT116 cells stably expressing shRNA against PRL-3 (shPRL-3) or control shRNA (shCon) were cultured and analysed as in (a). (d) Cell lysates from (a) were immunoprecipitated with a configuration-specific anti-Rheb-GTP antibody and analysed by immunoblotting with the indicated antibodies. (e) Cell lysates from (b) were analysed as in (d). (f) Proposed signalling pathway for PRL-3 to mTOR substrates, 4EBP1 and p70S6K. Representative full immunoblots for the cropped images presented here are provided in the Supplementary Information.