Luc2p nf κb re hygro vector

HEK293T and A549 cells were cultured in 24-well plates, and co-transfected with the pGL4.32 [luc2P/NF-κB-RE/Hygro] vector (Promega) and the pRL-TK Renilla Luciferase control reporter vector (Promega). At 24 h after transfection by PEI, the cells were lysed and the luciferase activity was measured with a GloMax 20/20 luminometer (Promega), using the Dual-Luciferase Reporter Assay System (Promega). At 18 h after transfection, TNF-α (10 ng/ml) or IL-1β (1 ng/ml) was added to the medium. The cultures were incubated further for 6 h and then the cells were analyzed.

NF-κB luciferase reporter gene cells were generated by stably transfecting SH-SY5Y cells with the pGL4.32[luc2P/NF-κB-RE/Hygro] vector (Promega, Charbonnières les bains, France) containing five copies of an NF-κB response element that drives transcription of the luciferase reporter gene luc2P and a mammalian selectable marker for hygromycin resistance. After hygromycin selection (200 µg/mL), surviving cells were transfected with a control vector containing β-galactosidase with a cytomegalovirus promoter. 48 h after transfection, serum starved cells were treated 4 h with resistin (200 ng/ml), palmitic acid (200 µM) or DHA (20 µM) then luciferase and β-galactosidase activity were measured using the Mithras LB 940 apparatus (Berthold technologies, Bad Wildbad, Germany) and the Dual-Light Chemiluminescent Reporter Gene Assay System (Applied Biosystems, llkirch, France) according to the manufacturer’s instructions. Luciferase activity was normalized by β-galactosidase activity. All transfections were performed using Lipofectamine 2000 (Invitrogen, llkirch, France) according to the manufacturer’s instructions.

RAW264.7 cells were seeded in a 24-well plate at a density of 3 × 10⁵ cells/well. Then, 0.8 µg of pGL4.32 (luc2P/NF-κB-RE/Hygro) vector (Promega, Madison, WI, USA) was transfected into cells using the lipofectamine 2000 reagent as described previously [24 (link)]. Cells were exposed to fargesin (5, 10, and 25 μM) in advance for 2 h and then incubated with LPS (2 µg/mL) for an additional 12 h. A dual luciferase reporter assay kit was used to measure luciferase activity. Renilla luciferase was used to normalize the transfection efficiency.