Transfection and Cell Line Generation

TREx293 cells, that stably express the Tet repressor (Thermo Fisher Scientific), were transfected with the expression vector pcDNA4TO-YAP1 and selected with Zeocin (Invitrogen). The cell lines TREx293, HEK293T, LNCaP, A427, HeLa and A549 were cultivated in DMEM or RPMI with 10% FCS at 37°C under 5% CO₂ concentration. Colon cancer cell lines HCT116 p53^+/+ and HCT116 p53^−/− were obtained from Thorsten Stiewe (University Marburg, Germany) and cultivated in DMEM [40 (link)]. The cells were transfected at a confluency of 60–80% in serum free media (GIBCO) with 4 or 10 µg DNA (3.5 cm, 10 cm plates respectively). HEK293T and TREx293 cells were transfected using PEI. LNCaP were transfected with Lipofectamin (Invitrogen). A549 were transfected using Turbofect (Thermo Fisher Scientific). A427 and HCT116 cells were transfected using X-tremGENE HP (Roche). HeLa were transfected using JetPEI (Polyplus) according to the manufacturer’s instructions. For the colony formation assay, the selection was performed using G418 (Biochrom) or Zeocin (Invitrogen) and colonies were stained with Giemsa (Fluka). Primary human hepatocellular carcinomas and matching normal tissue samples were obtained from patients of the University of Halle-Wittenberg and were previously described [59 (link)]. The local committee of medical ethics approved the use and all patients gave their consent.