Nf κb luciferase reporter

RAW 264.7 cells were plated at 1 × 10⁵ cells/well on 24-well culture plate and transiently transfected with NF-κB-luciferase reporter (Affymetrix Inc., Santa Clara, CA, USA) or pNF-κB-luciferase reporter (Stratagene California, La Jolla, CA, USA) using Lipofectamine^® LTX & PLUS in OPTI-MEM media (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instruction. The transfected cells were treated with LPS (1 μg/mL) in the absence or presence of various concentrations of TECA, AST and TECA + AST for 24 hours. Reporter gene activity was assayed using the luciferase assay kit (Promega Co., Madison, WI, USA), measured by a luminescence counter (Wallac Victor2 1420; PerkinElmer Inc., Waltham, MA, USA).

A 366 bp fragment of DR5 3′ UTR (863–1,228 nt) was amplified by PCR and cloned into the vector pMIR reporter (Promega). To investigate whether this UTR was directly targeted by miR-133a and miR-30b, the predicted target site was mutated by site-directed mutagenesis. These reporter vectors were referred as WT and Mu vectors, respectively. Following previous studies,⁴⁰ (link) the WT or Mu vector and miRNAs were co-transfected into U87, M059J, and A172 cells, and each experiment was repeated for three times. Luciferase activity was assayed 28–30 hr after cotransfection using the dual luciferase reporter assay system (Promega). The Renilla luciferase activities were used for normalization. For the NF-κB luciferase activity assays, the NF-κB luciferase reporter was purchased from Affymetrix, and luciferase activities were determined as described earlier.