White walled 96 well plate

Cells were plated in triplicate at 2 × 10³ per well in white-walled 96-well plates (Becton Dickinson) for 24 hours and then were treated with the IKK inhibitor and/or cisplatin for additional 48 hours. Caspase-3/7 activity was measured using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's instructions. Caspase-Glo 3/7 assay uses a caspase-3/7 tetrapeptide DEVD substrate that produces a luminescent signal on cleavage. Relative light units were measured on an Lmax Microplate Luminometer (Molecular Devices).

For the dual luciferase replicons, the commercial Nano-Glo Dual-Luciferase Reporter Assay System (Promega) was used. In brief, 40 µl of cell lysate were transferred from the six-well plate to a white walled 96-well plate (Falcon) in duplicates. Next, 40 µl of ONE-Glo EX Reagent (Promega) were added, mixed and the reaction was incubated for 10 min at room temperature before measurement of the firefly luciferase signal. Then, firefly activity was quenched for 10 min using 40 µl of NanoDLR Stop& Glo buffer (Promega) before determining the Nano luciferase signal. Non-transfected cells were used to determine the background signal. Measurements were performed using a Mitras LB940 plate reader (Berthold).