Mw 25k

HEK293T and 786-O cell lines were purchased from the American Type Culture Collection and cultured as previously described [11 ]. HEK293T cells were transfected with the expression plasmid, using polyethyleneimine (PEI) (MW-25K, Polyscience Inc., Warrington, PA, USA), plasmid DNA (μg):PEI (μg) = 1:3. Retroviral infections were performed as described previously [28 (link)]. In brief, 786-O cells were incubated in retrovirus-containing culture medium for 2 days and selected using puromycin (5 μg/ml) for 1 week. Additionally, siRNA was transfected into 786-O cells, using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) per the manufacturer’s instructions. At 2 days post transfection, cells were harvested and lysed.

HEK293T, 786-O, K562, RCC4, and U-2 OS cells (American Type Culture Collection, ATCC) were cultured in DMEM (Fujifilm Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL), and streptomycin (100 μg/mL). C2C12 cells were a gift from Dr. Masafumi Kuzuya (Nagoya University, Japan). C2C12 cells (RCB0987, lot #41) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. MCF10A cells were cultured in DMEM/Ham’s F-12 (Fujifilm Wako Pure Chemical Corporation) supplemented with 5% FBS, 100 units/mL penicillin G (Nakalai, Japan), 100 μg/mL streptomycin (Nakalai, Japan), 20 ng/mL epidermal growth factor (Novus Biologicals, USA), 10 μg/mL insulin (Roche, Germany), 1 ng/mL cholera toxin (Bio Academia, Japan), and 1 μg/mL hydrocortisone (Tokyo Chemical Industry). Confluent C2C12 cells on gelatin-coated dishes were differentiated into myotubes by replacing 10% fetal calf serum (FCS) with 2% horse serum. The medium was changed every day for the indicated periods. MG132 (2 μM) was added to the culture medium for the indicated period. HEK293T and C2C12 cells were transfected with expression plasmids using polyethyleneimine (MW-25 K; Polysciences Inc., USA) at a mass ratio of 1:3.