Torin1

U2OS cells were obtained from ATCC. U2OS GFP‐LC3 and U2OS mRFP‐EGFP‐LC3 stable cell lines are a kind gift from Tassula Proikas‐Cezanne (Eberhard Karls University Tübingen, Germany) and Jeff MacKeigan (Van Andel Research Institute, MI, United States), respectively. All cells were grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, BioWhittaker) supplemented with 10% fetal calf serum (FCS), 2 mM l‐glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, referred to as CM. For starvation in nutrient‐depleted medium, the cells were incubated in EBSS (Gibco, Life Technologies) for 2 h. BafA1 (AH Diagnostics) was used at 100 nM and Torin1 (R&D Systems) at 50 nM.
Blood components (buffy coats) from healthy blood donors were obtained from the local blood bank (Ullevål University Hospital, Oslo, Norway) according to the guidelines of the local blood bank approved by the Norwegian Regional Committee for Medical Research Ethics. Mononuclear cells were isolated through density gradient centrifugation by using Lymphoprep (Axis Shield). MDDCs were generated through culture for 6 days in RPMI media containing 100 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF; Immunotools) and 20 ng/ml IL‐4 (Invitrogen, Life Technologies) supplemented with 10% FCS, 2 mM l‐glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.