Puromycin puro

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 Plasmocin^TM (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.

HeLa cells (1 × 10⁶) were transduced with the control (scramble) or human NUP153-specific shRNA lentivirus particles overnight at 37 °C followed by selection for 48 h  in medium containing Puromycin (Puro, 2 μg/ml) (Sigma-Aldrich). To collect cells at the basal (minus EGF) IEG state, cells were pre-cultured in DMEM supplemented with 0.1% FBS (Sigma-Aldrich) for 24 h, followed by treatment with EGF (50 ng/ml) (Sigma-Aldrich, E9644) for 15, 30, 60, 90, and 120 min. For the rescue experiments, HeLa cells were transfected with control (scramble) or NUP153-specific shRNA vectors along with FLAG-hNUP153 expression vector using Xfect transfection reagent (Clontech) according to the manufacturer’s instructions. At the 16 h time point, culture medium was replaced with Puro (2 μg/ml) containing medium and cells were incubated in this medium for 24 h, followed by incubation in Puro-free medium for another 24 h. To induce IEG transcription, cells were subjected to EGF treatment as described above.