NFκB Transcriptional Regulation Assay

Ovcar3 and Ovcar8 cells subjected to scrambled or Caspase 8 shRNA knockdown were first selected with puromycin as described before^{4 (link)}. After selection, cells were transduced with a lentiviral vector containing an NFκB transcriptional regulatory element, using the Cignal Lenti Reporter System (CLS-013L), according to the manufacturer’s specifications and allowed 72 h for maximum vector expression. Transient reporter assays were subsequently performed for 18 h. Briefly, cells were plated in 96-well plates at a density of 10,000 cells/well. After overnight attachment, cells were exposed to serum starvation medium containing 0.5% fetal bovine serum for 24 h. Drugs or IKKβ inhibitor IV (EMD Biosciences) were added for 1 h after which TNFα (300-01A, PeproTech) was added to stimulate NFκB activity for 18 h. Control wells received vehicle alone. Luciferase activity was measured using the Luciferase Assay System (E4030, Promega) according to the manufacturer’s instructions and a Spectramax M5 plate reader (Molecular Devices). Luciferase units were normalized to viable cell number, obtained by XTT assay, on duplicate assay plates. Experiments included 3–6 replicate samples per point and were repeated at least three times.