Transient Transfection and Luciferase Assay

Transient transfection of reporter plasmids and luciferase assays was performed as described previously^{4 (link), 80 (link)}. 1 × 10⁵ cells were seeded per well in 24-well plates 24 h before transfection. Lipofectamine^TM LTX and Plus^TM reagents (Invitrogen) were used for DNA transfection, according to the manufacturer’s instructions. 400 ng of pGL3p-N3Int2 was transfected along with or without 400 ng of pBABE-bla-ZEB1, pBABE-bla-ZEB2 of pBABE-bla (empty vector control). 5 ng of phRL-SV40-renilla luciferase vector (Promega) was co-transfected to calibrate the variation of transfection efficiencies among wells. Cells were incubated in the presence or absence of 1 µg/ml DOX to induce ICN1 in cells expressing ICN1^TetOn for 48 h before cell lysis. Alternatively, 5 ng/ml TGFβ1 was added at 24 h after transfection and incubated for an additional 48 h before cell lysis. Luciferase activities were determined using Dual-Luciferase^TM Reporter Assay system (Promega) and ORION Microplate Luminometer (Berthold Detection Systems, USA, Oak Ridge, TN). The mean of firefly luciferase activity was normalized with the co-transfected renilla luciferase activity. Transfection was carried out at least three times, and variation between experiments was not greater than 15%.