Cells were seeded on 35-mm polylysine-coated glass bottom plates, serum-starved (20 hours), pretreated with 100 nM torin1, and treated with 2.5 mM AICAR (5 hours). Cell viability was assessed by adding ReadyProbes reagents (Thermo Fisher Scientific; catalog no. R37609) for the last 15 min of incubation, according to the manufacturers’ instructions. NucBlue Live reagent (Hoechst 33342) stains the nuclei of all cells, whereas NucGreen Dead stains the nuclei of dead cells with compromised plasma membranes. Images were collected on an inverted epifluorescence microscope (Nikon TE2000E) equipped with a Photometrics CoolSnap HQ camera and analyzed using ImageJ (National Institutes of Health).

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