Centro lb960 dual luminometer

The MMP-9 promoter reporters, wild-type pMMP9-Luc(−925/+13), and disrupted NF-κB binding site pMMP9-Luc(−925/+13)mtNFκB are described elsewhere31 (link). MDA-MB-231 cells cultured on 12-well plates were transfected with 0.5 μg of the MMP-9 promoter reporter constructs, along with 50 ng of the pRL-null plasmid encoding Renilla luciferase to monitor transfection efficiency. At 48 h post-transfection, the cells were treated with 10 ng/mL TNFα in the presence or absence of 10 μM DPP23. After 8 h, the cells were collected, and the firefly luciferase activities were measured as previously described31 (link). Luminescence was measured using a Centro LB960 dual luminometer (Berthold Technologies).

The generation of the MMP9 promoter construct pMMP9-Luc(−925/+13) and the mutated construct for EGR-1 binding site, pMMP9-Luc(−925/+13)mtEgr1, was described elsewhere [28 (link)]. MDA-MB-231 cells cultured on 12-well plates were transfected with 0.1 μg of MMP9 promoter-reporter constructs along with 25 ng of the pRL-null plasmid encoding Renilla luciferase to monitor transfection efficiency. At 48 h post-transfection, cells were pretreated with either vehicle (DMSO) or DK4023 (25 or 50 μM). After 30 min, cells were treated with either vehicle (PBS) or TNFα (10 ng/mL). After 8 h, cells were collected and the firefly luciferase activity was measured using a Centro LB960 dual luminometer (Berthold Technologies) as previously described [28 (link)].