Pentr4 v5

CtBP DD cells, 3% O2, 5% CO2 and 92% N (O2/CO2 incubator MCO-5M-PE, Panasonic Healthcare Co. Ltd, Japan). 2-deoxyglucose (2-DG), Dimethyloxalylglycine (DMOG) and 2keto-4-methylthio-2-oxo butyrate (MTOB) were from Sigma-Aldrich, UK). siRNA (Ambion, Applied Biosystems, UK) targeting both CtBP1 and CtBP2 (CTBPsi) [54] , HIF1A (S6541) and silencer negative control #1 (NSsi) were transfected using INTERFERin (PolyplusTransfection, France). his-CtBP2 and his-CtBP2 G189A constructs [55] were transfected into cells using FugeneHD (Promega, UK), and single colonies expanded under G418 selection. To generate the IND CtBP DD and IND CtBP DDM vector, CtBP DD (CtBP2[104-361]) and CtBP DDM (CtBP2[104-361]R147L,R169L) [46] were cloned into pENTR4-V5 (Addgene #17425) and then pLentiCMVtighthygroDEST (Addgene #26433) [56] , which was co-transfected into HEK-T cells with pCMVdR8.91 and pMD2.G packaging vectors to generate lentiviral particles. The generated lentiviral particles were transduced into MCF-7, which had previously been transduced with particles generated using pLentiCMVrtTA3 Blast (Addgene #26429) controller vector. Transduced cells were selected with blasticidin (10 μg/mL) and hygromycin (200 μg/mL). Doxycycline was from Clontech, USA.