Rapamycin

Embryos were collected every 3 h on apple juice agar plates, and incubated at 25°C for 24 h; hatching first instar larvae were transferred into 2.0 mL Eppendorf tubes with food containing 0.05% (v/v) ethanol (vehicle control), 1 μM rapamycin (AG Scientific), 0.2 or 2 μM Wortmannin (AG Scientific; R-1018) or 5 mM 3-methyladenine (Calbioche^® m, Merck Millipore, 189490), and dissected as third instar larvae. 10 μM Chloroquine was added to primary cultures for 2 h before fixation and staining with anti-mCherry. For Westerns, 5 third instar larval brains or 3 heads per treatment per lane were collected, frozen on dry ice, and ground with a pestle in RIPA lysis buffer (Thermo Fisher Scientific) with protease inhibitor cocktail. Rabbit anti-Bchs [for details see Lim and Kraut (2009) (link)] or Rabbit anti-pS6K (Cell Signaling) were diluted 1:6000 and 1:1000, respectively, incubated on blots overnight at 4C, and detected with HRP-coupled secondary and SuperSignal West Pico plus reagent (Thermo Fisher Scientific).

Wild type S. cerevisiae strain L5366 (wild type, MATa/α ura3-52/ura3-52 in the Σ1278b strain background^{41 (link)},.pngt from Dr. A. H. Limper) and the double deletion strain L5366 Δacb1 (Δacb1::URA3/Δacb1::URA3) as well as Hho1 GFP-tagged or mCherry-tagged variants thereof (L5366 mCherry and L5366 Δacb1 GFP) were used. HHO1 codes for the Histone H1 protein. Analyses of autophagy in yeast were carried out in BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) and respective ∆acb1 mutant obtained from EUROSCARF. Strains were grown at 28 °C on synthetic minimal medium containing 0.17% yeast nitrogen base (Difco), 0.5% (NH₄)₂SO₄ and 30 mg/L of all amino acids (except 80 mg/L histidine, 120 mg/L lysine, and 200 mg/L leucine), 30 mg/L adenine and 320 mg/L uracil with 2% glucose. For chronological aging, cells were inoculated to OD_600nm 0.1 from fresh overnight cultures and grown at 28 °C. Where indicated, cultures were supplemented with 40 nM rapamycin (AG Scientific; 1 mg/mL stock in DMSO) at the time point of inoculation. For nitrogen starvation, cells where grown to OD_600nm 1 on synthetic minimal medium and then transferred to medium without amino acids and (NH₄)₂SO₄.

FA was supplied by Sigma‐Aldrich (St Louis, MO., USA); rapamycin was supplied by AG Scientific, Inc. (San Diego, CA., USA); ELISA kits were purchased from R&D systems (Minneapolis, MN., USA); oxidative stress detection kits were purchased from Jiancheng Bioengineering Institute (NanJing, China); oil red O stain was obtained from Sigma‐Aldrich; Lillie‐Mayer's haematoxylin and eosin (H&E) stain was obtained from Cosmo Bio Company (Tokyo, Japan); Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), cell lysis buffer (Cell signalling technology, USA) and antibodies against α‐smooth muscle actin (SMA), OPN, mTOR, phosphorylated (p)‐mTOR, p70S6K and phosphorylated (p)‐p70S6K were obtained from Cell Signaling (Danvers, MA., USA).