Dual glo luciferase reporter kit

Cells were transfected with the NF-κB Cignal reporter vector (CCS013L, QIAGEN), containing the firefly luciferase gene controlled by an NF-κB responsive promoter and a vector encoding the renilla gene under a constitutive promoter. After 24 hours, media was replaced with fresh media containing 10μM I-BET151 or vehicle control. After the indicated length of time, luciferase and renilla activity were detected using the Dual-Glo Luciferase Reporter kit as directed by the manufacture (Promega, Fitchburg, WI).

On day 7 of differentiation SGBS adipocytes were nucleofected with a NFκB Firefly luciferase reporter vector containing five copies of an NFκB response element that drives transcription of the Firefly luciferase reporter gene and Renilla luciferase control reporter vector using the Neon Transfection System (Thermo Fisher Scientific, Darmstadt, Germany). First, adipocytes were trypsinized, counted and a cell solution of 600000 cells was centrifuged and resuspended in 105 μl Resuspension Buffer B of the Neon Transfection Kit (100 μl). The nucleofection was performed with 7.5 μg of the NFκB Firefly luciferase reporter vector^{55 (link), 56 (link)} and with 0.75 μg of the Renilla luciferase control reporter vector (pRL-TK; Promega, Heidelberg) at 1400 Volt with three electric shocks for 10 ms each. Then, 75000 cells were seeded into one well of a 12-well plate. After 24 hours the adipocytes were treated for additional 24 hours with either TRAIL (30 ng/ml), TNF-α (30 ng/ml) as a positive control or vehicle as a negative control. The cell lysates were harvested by scraping and the luciferase activities were measured with the Dual-Glo Luciferase Reporter Kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Luminescence was recorded in a Multimode Microplate Reader (Mithras LB 940, Berthold, Bad Wildbad, Germany).