HEK293T cells (fetal; ATCC), HeLa cells (female; ATCC), and HeLa cells stably expressing GFP-LC3B (a gift from Dr. Chungho Kim, Korea University, Seoul, Korea) were cultured in Dulbecco′s modified Eagle′s medium (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). To prevent mycoplasma contamination, all the cultured cells were regularly treated with PlasmocinTM (Invivogen) and examined using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza).
Where indicated, the cells were treated with rapamycin (Rapa; 500 nM for HEK293T cells and 100 μM for HeLa cells; Enzo Life Science) predissolved in DMSO (BioShop) with or without 30 μM chloroquine (CQ; Sigma-Aldrich) for 12 h. In addition, the cells were treated with 10 mM 3-MA (Sigma-Aldrich), 100 nM Baf A1 (Calbiochem), 1 μg/ml puromycin (Puro; Sigma-Aldrich), or 50 μg/ml cycloheximide (CHX; Sigma-Aldrich) for 12 h.
Where indicated, the cells were treated with rapamycin (Rapa; 500 nM for HEK293T cells and 100 μM for HeLa cells; Enzo Life Science) predissolved in DMSO (BioShop) with or without 30 μM chloroquine (CQ; Sigma-Aldrich) for 12 h. In addition, the cells were treated with 10 mM 3-MA (Sigma-Aldrich), 100 nM Baf A1 (Calbiochem), 1 μg/ml puromycin (Puro; Sigma-Aldrich), or 50 μg/ml cycloheximide (CHX; Sigma-Aldrich) for 12 h.