Geneticin g418

Human bladder carcinoma EJ cells [40 (link)] were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning, 10-013-CV) containing 10% Fetal Bovine Serum (FBS, Atlanta Biologics), 100 μg/ml of Hygromycin (Sigma, H3274), and 200 μg/ml Geneticin G418 (Teknova, G5005). P53 knock-out EJ cells (EJ p53 off) and p53 expressing EJ cells (EJ p53 on) were established respectively by the presence and the absence of 1 μg/ml doxycycline (Sigma, D9891) in the culture media. Human colorectal carcinoma HCT116 cells [41 (link)] were cultured in DMEM containing 10% FBS. Both EJ and HCT 116 cells were maintained at 5% CO₂ and 37°C. All the human cell lines and experimental protocols were approved by Harvard Institute Review Board and carried out in accordance with the relevant guidelines and regulations. The datasets generated during the current study are available from the corresponding author on reasonable request.

Huh7.5, Huh7 and 293 T cells were generously provided by Charles Rice (Rockefeller University, NY) and Huh7.5.1 cells as a kind gift from Frank Chisari (The Scripps Research Institute, CA). Huh7.5 cells expressing a shRNA against either human CypA (Huh7.5-shRNA CypA) or an irrelevant target (Huh7.5-shRNA irrel) were graciously provided by Thomas von Hahn and Sandra Ciesek (Hannover Medical School, Germany; University of Duisburg-Essen, Germany) (von Hahn et al., 2012 (link)). Huh7 cells were obtained from the American Tissue Culture Collection (ATCC) and Hep56.1D cells from CLS Cell Lines Service GmbH (Eppelheim, Germany). The Hep56.1D-derived "Clone 8" cells were generated as described below. All cells have been authenticated and are clear of mycoplasma contamination. All cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher) supplemented with 10% (v/v) fetal bovine serum (FBS) (Omega Scientific). To select for the shRNA, Huh7.5-shRNA CypA and Huh7.5-shRNA irrel cells were maintained under blasticidin selection at 5 ug/mL (BioVision). Cells transduced with lentivirus expressing the different CypA orthologs (see below) were selected for with Geneticin (G418) (Teknova) at 750 µg/mL. All infections were performed in the absence of antibiotics. Cells were maintained at 37°C in a 5% (v/v) CO₂, 20% (v/v) O₂ environment.