Antibodies and Reagents
Torin 1 was purchased from BioCat and Bafilomycin A1 from AlfaAesa. Antibodies were purchased from the following sources and used at the indicated dilution: Cell Signaling: Raptor (2280, 1:1000), Rictor (2114, 1:1000); Progen: p62 (GP62-C, 1:2000 dilution); Novus Biologicals: LC3B (NB100-2220, 1:2000); Sigma: Actin (A0483, 1:10,000).
U2OS Autophagy LC3 HiBiT Reporter Cell Line (Promega) was maintained in McCoy’s 5A media containing 10% FBS, 1% P/S, ʟ-Glutamine and 250 μg/ml G418 (Gibco).
siRNA transfections were performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Human Dicer-siRNA for Raptor, Rictor and non-targeting control were obtained from Integrated DNA Technologies (IDT).
Cell Lysates and Western Blot Analyses
Cells were washed in ice-cold PBS and lysed in M-PER lysis buffer (Thermo Fisher Scientific) containing protease inhibitors (Complete Mini; Roche), 5mM NaF, 1mM Na3VO4 and phosphatase inhibitors cocktail (PhosSTOP; Roche) for 10 minutes at 4°C. Lysates were cleared by centrifugation at 16,000 × g for 10 minutes. Cleared lysates were diluted in 4Χ Roti Load loading buffer (Roth-Chemie GmbH) and denatured at 95°C for 10 minutes. Protein samples were loaded on a 5–20% gradient gel (Bio-Rad), transferred to PVDF membranes (Millipore) and incubated with the indicated antibodies.
Autophagy LC3 HiBiT Reporter Assays
Cells were seeded (8,000 cells/well) in white 96 well plates (Corning). Twenty-four hours later, cells were washed twice with PBS and media was changed to 50μl of normal media or starvation (HBSS) media containing the different treatments and corresponding controls for 3 hours. Autophagy LC3 HiBiT Reporter levels were measured using the Nano-Glo® HiBiT Lytic Reagent (Promega) according to manufacturer’s instructions.