Autolumat lb953 luminometer

DNA plasmid constructs were transfected into cells in six-well plates (Corning,), using Lipofectamine 2000 for human cells. Cells were exposed to serum-free medium containing 1 μg DNA of different hiNOS promoter constructs with 10 μg of liposomes for 6 h, washed, and replaced with medium supplemented with 5% calf serum. Cells were lysed with Dual-Glo luciferase Assay System (Promega). Luciferase activity was examined by AutoLumat LB 953 luminometer (Berthold).
Control siRNA (sc-37007) and siRNA against p300 (sc-29431) were obtained from Santa Cruz Biotechnology, and cells were transfected according to the manufacturer's instruction. About 2 × 10⁶ cells were seeded on 6 well cell culture plates one prior to the day of transfection and grew to 60–80% confluent. For each transfection, 50 pmols control siRNA or p300 siRNA were added in siRNA transfection medium. Cells were treated with siRNA medium for 5 hours. The medium were aspirated and replaced with normal growth medium. The sequence of p300 siRNA (sc-29431) is: CCCCUCCUCUUCAGCACCA. The p300 siRNA (sc-29431) was validated in A549 cells by Western Blot (S1 Fig).

Luciferase assays were carried out using Luciferase Assay System (Promega, Madison, USA) and AutoLumat LB953 luminometer (Berthold, Wildbad, Germany) according to the manufacturer's protocols. In brief, 3×10⁴ cells seeded in a 16-mm tissue culture dish were transfected with 1 ug of luciferase reporter plasmids and 0.25 ug of pSV-β-galactosidase control plasmid vector. Cell lysates were prepared 48 hours after transfection by adding 100 ul of reporter lysis buffer and their luciferase activities were measured. The pGL3-Promoter plasmid containing SV40 promoter was used as a positive control. The luciferase activity of the pGL3-Promoter plasmid in each cell line was considered to be 1, and the relative luciferase activity was calculated. All luciferase assays were performed in triplicate.

For luciferase reporter experiments, sequences of the ZEB1 promoter were constructed into a pGL3 vector (Promega, Madison, WI, USA). Then, pcDNA3.1, pcDNA3.1/YAP1, or pcDNA3.1/TAZ1 was respectively transfected into the HEK293T cells together with pGL3-ZEB1 promoter, using Lipofectamine 2000 (Invitrogen). 48 h after transfection, the luciferase activity was evaluated by the use of an AutoLumat LB953 luminometer (Berthold).