Restoration of membrane potential in senescent cells promotes cilia elongation. (a) Resting and maximum membrane potential (maximum achieved by incubation for 5 min with 80 mM potassium gluconate) were measured in control and senescent (20 Gy irradiation; Sen (IR)) fibroblasts using DiSBAC2(3) dye. Depolarization is indicated by increased fluorescence. Data are represented relative to maximum. (b) Senescent cells treated as in a but in the presence or absence of 100 µM pinacidil overnight. (c and d) Senescent fibroblasts were treated with pinacidil overnight in serum-free medium. Cells were fixed and stained for acetylated tubulin (c). Percentage of cells with cilia and cilia length were quantified (d). (e) Senescent fibroblasts were starved of serum overnight and amino acids for 1 h in the presence or absence of pinacidil as indicated, lysed, and analyzed by immunoblot for mTORC1 activity. (f) Senescent fibroblasts were starved of serum overnight and amino acids for 1 h in the presence or absence of 50 µM autophagy inhibitor chloroquine (CQ), 10 µM Akt inhibitor, or both inhibitors as indicated. Cells were lysed and immunoblotted. Error bars represent SEM; all experiments, n = 3 (for immunofluorescence, at least five fields of view imaged per experimental repeat). Student’s t test performed between groups: **, P < 0.01; ***, P < 0.001; NS, not significant. Bars: (main) 10 µm; (insets) 5 µm.
