Culturing Human Cell Lines and Drug Treatments

Human foreskin fibroblasts (HFF-1) from the American Type Culture Collection (ATCC, Manassas, VA), hTERT-immortalized retinal pigment epithelial cells (hTERT-RPE1; ATCC), and human embryonic kidney cells, 293FT (HEK-293FT; Invitrogen) were cultured as previously described (Kalab et al., 2006 (link); Hasegawa et al., 2013 (link)). Human colorectal carcinoma HCT116 cells were cultured in DMEM with 10% fetal bovine serum (FBS). Normal human fibroblasts CRL1474 (ATCC) were cultured in MEM with 15% FBS and normal lung fibroblasts Wi38 (ATCC) in MEM with 10% FBS. Media of cells expressing RCC1-V5 was supplemented with 10 μg/ml blasticidin (Invitrogen). All cells were grown in a 37°C humidified incubator with 5% CO₂ and in media supplemented with 100 U/ml penicillin and 10 μg/ml streptomycin. Cells were treated with Importazole (IPZ; Sigma-Aldrich, St. Louis, MO; final concentration 40 μM) and KPT-330 (Karyopharm, Newton, MA; final concentration 500 nM) by supplementing their complete cell culture with aliquots of 10 mM solutions of the drugs in dimethyl sulfoxide.