Tet repressor

The A549, H322 and A427 human cell lines (ATCC) were maintained in DMEM F12 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (PAA, Austria), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine at 37 °C in a 5% CO₂, 95% O₂. NHBEC were obtained from Clonetics (Belgium) and grown in BEGM. TREx293 cells, which stably express the Tet repressor (Thermo Fisher, USA), were transfected with the expression vector pcDNA4TO and selected with Zeocin (Invitrogen). HEK293T and TREx293 were transfected using polyethylenimine. All patients gave a written consent at initial clinical investigation. Consent has been obtained to publish in an online-access publication, if the information could lead to the identification of a study participant. The study and experimental protocols were approved by the ethical committee of the Medical Faculty of University Halle-Wittenberg, Germany^{2 (link)}. All experiments were performed in accordance with relevant guidelines and regulations.

AATK coding sequence was kindly provided by Ma and Rubin [16 (link)] and cloned into, pEYFP-C2 (Clontech) and pCMVTag1 (Flag; Agilent). The ATP binding pocket of AATK was mutated (Ala>Lys) by site-directed mutagenesis (Agilent). DNA of AATK wt or AATK KD were cloned into pcDNATo/Myc/His vector (Thermo Fisher Scientific). TREx293 cells, that stably express the Tet repressor (Thermo Fisher Scientific), were transfected with the expression vector pcDNA4TO-AATK wt or pcDNA4TO-AATK KD and selected with Zeocin (Thermo Fisher Scientific). The cells were cultivated in DMEM with 10% tetracycline-free serum (Biochrom) and 1% penicillin and streptomycin (GIBCO). The selection of the clones was performed using blasticidin (5 µg/ml, Roth) and Zeocin (500 µg/ml, Thermo Fisher Scientific). The induction of AATK wt or AATK KD was performed using doxycycline (2 µg/ml, Thermo Fisher Scientific).

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.