Cmv renilla luciferase vector

For dual luciferase reporter gene assays, cells grown in 6-well plates were cotransfected with 2 μg of the firefly luciferase vector containing miR-29c promoter and 10 ng of CMV-renilla luciferase vector (Promega, Madison, WI, USA) using Dharmafect Duo transfection reagent (Dharmacon, Lafayette, CO). For chromatin immunoprecipitation (ChIP) assays, cells were cross-linked, sonicated, and immunoprecipitated with an anti-RNA Polymerase II antibody using Magna ChIP kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocols. Normal mouse IgG was used as a negative control.

One day prior to transfection, 3 × 10⁵ cells were seeded into one well of a 12-well tissue-culture plate (BD Biosciences, San Jose, CA). After 12 h, co-transfections were conducted using the transfection vector mixture (total 333 ng) containing NF-κB RE luc reporter construct (300 ng, a kind gift from Dr. Lyndon F Cooper) or PGL Basic reporter construct (Promega). CMV/Renilla luciferase vector (33 ng, Promega) was used as transfection efficiency control. 0.6 μl of transfection agent Lipofectamine 2000 (Invitrogen) was added to the mixture, and co-transfections were conducted according to the manufacturer's instruction. Sixteen hours later, the cells were treated with TPCA-1 at 10 µM for 30 min followed by addition of DPP (500 ng/ml). After 48 h, cell lysates were prepared using passive lysis buffer (Promega). Luciferase activities in the lysates were measured using a dual luciferase assay system (Promega) with a plate reader (Synergy 2, BIOTEK).