Plustm

PFF cells were maintained in the culture media, as described previously [24 (link)]. Cells were incubated at 37 °C in 5% CO₂ to reach 80% confluence for transfection. PFF cells were cultured in 24-well plates and transfected with 0.75 μg of either P1-P6, P5-1/2/3, or P5-9T/10T/11T with pRL-TK vector containing Renilla luciferase. Furthermore, co-transfection was also carried out on PFF cells by co-transfecting P5-9T/10T/11T and C/EBPα. The transfection method was operated according to the instructions of lipofectamine TM LTX and PLUSTM (Invitrogen, Thermo Fisher Scientific Inc., Carlsbad, CA, USA). Luciferase activity was measured 48 h later using the Dual-Glo luciferase assay (Promega, Madison, WI, USA). The activities of different promoter fragments were expressed by detecting the ratio of firefly luciferase activity to Renilla luciferase activity [26 (link)], which allowed for the evaluation of which fragment was a IGF1 core promoter.

The 3′UTR of the DNMT1 gene, which contains a miR-148a target site, was cloned into a pEZX-MT05 vector (GeneCopoeia) containing SV40 promoter-driven Gaussia luciferase and CMV promoter-driven secreted alkaline phosphatase (SEAP) genes. A mutant DNMT1 3′UTR was also constructed. 293T cells were seeded in 24-well plates for 1 day and then co-transfected with pEZX-miR-148a or pEZX-miR-control and pEZX-DNMT1-3′UTR-WT or transfected with pEZX-DNMT1-3'UTR-MUT (3:1) using Lipofectamine LTX and PLUS^TM (Invitrogen). At 48 h post-transfection, the supernatants were collected and the luciferase activities were measured on a Centro LB 960 microplate luminometer (Berthold Technologies) using a Secrete-Pair Dual Luminescence Assay kit (GeneCopoeia) according to the manufacturer’s protocols.

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%.