Pgl4.75 hrluc cmv vector

The NF-κB reporter plasmid, pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega, Madison, WI, USA), contains five copies of an NF-κB response element (NF-κB-RE) that drives the transcription of the firefly luciferase reporter gene. Briefly, 3 million PEDSV.15 cells in 0.4 mL ice-cold PBS were electroporated with 5 µg pGL4.32[luc2P/NF-κB-RE/Hygro] and 200 ng pGL4.75[hRluc/CMV] vector (Promega, Madison, WI, USA) reporter plasmids. The cells were seeded in 96-well plates (3 × 10⁴ cells/100 µL/well), and after overnight incubation, the cells were treated with inhibitors or DMSO for 30 min and stimulated for six hours. Cell extracts were prepared in 25 μL of 1× passive lysis buffer per well (Biotium, Fremont, CA, USA). The samples were assayed for firefly and Renilla luciferase activities using the Firefly & Renilla Luciferase Single Tube Assay Kit (Biotium, Fremont, CA, USA) and a Centro LB 960 luminometer (Berthold Technologies, Bad Wildbad, Germany).

TOPflash or FOPflash reporter plasmids (2 μg, Upstate Signaling) were co-transfected with 2 μg pGL4.75 (hRluc/CMV) vector (Promega) into 2 Mio cells (Nucleofactor Kit, Amaxa, efficiency of transfection: 30%^{61 (link)}). Cells were cultured 3 days in 24-well plates and subsequently stimulated with compounds for 24 h. Reporter activity was measured by using the Dual Luciferase Assay System (Promega). TOPflash or FOPflash activity was normalized to Renilla luciferase activity of pGL4.75, an internal standard for transfection efficiency.