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.
