HCT116 cells (4.5 × 105) were transiently transfected with 1 μg of pGL3N2PR-2327/-99, serial dilution of pEGFP–hTERT, or empty pEGFP-C1 and 0.5 μg cytomegalovirus β-galactosidase (pCMV-β-Gal). A set of experiments were also conducted with a smaller NOTCH2 luciferase promoter reporter construct (pGL3N2PR-110) instead of the longer pGL3N2PR-2327/-99. In some experiments, cells were treated with the inhibitors XAV-939 (Selleck Chemicals LLC, Houston, TX, USA) or N-AI (EDM Millipore) at the indicated concentration 3 h before transfection. DMSO was employed as control. As positive control, HCT116 were also co-transfected with pCGN-HA-S33Y-β-catenin a plasmid known to activate the NOTCH2 promoter or with pcDNA3 as control plasmid. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent according to the manufacturer's instructions.
TERT-negative U2OS cells (3x105) were transiently transfected with 1.5 μg phTERTpromoterLuc, 1 μg pMT2TMyc, or empty pMT2T, 1 μg pMigRI-ICN2 or pMSCVpuro-ICN2, and 0.5 μg CMV- β-Gal by Lipofectamine 2000 (Invitrogen). Luciferase and β-Gal activities were quantified with the Bright-Glo luciferase assay and Beta-Glo system (Promega, Madison, WI, USA), respectively. Luciferase activity was normalized to β-galactosidase activity and the data are presented as luciferase activity compared with that of the control vector.
TERT-negative U2OS cells (3x105) were transiently transfected with 1.5 μg phTERTpromoterLuc, 1 μg pMT2TMyc, or empty pMT2T, 1 μg pMigRI-ICN2 or pMSCVpuro-ICN2, and 0.5 μg CMV- β-Gal by Lipofectamine 2000 (Invitrogen). Luciferase and β-Gal activities were quantified with the Bright-Glo luciferase assay and Beta-Glo system (Promega, Madison, WI, USA), respectively. Luciferase activity was normalized to β-galactosidase activity and the data are presented as luciferase activity compared with that of the control vector.