Cignal lenti nfκb reporter

CMV-Renilla luciferase lentivirus (Cignal Lenti CMV Renilla Control; Qiagen, Inc.) was used as an internal control for normalization, and NF-κB reporter lentivirus with firefly luciferase expression (Cignal Lenti NF-κB Reporter; Qiagen, Inc.) was used as the experimental reporter. The two lentiviruses were used to co-transfect the OCI-LY10 and TMD8 cells. Briefly, OCI-LY10 and TMD8 cells were resuspended in 0.5 ml lentivirus-containing medium (containing 250 µl Cignal Lenti NF-κB Reporter and 250 µl Cignal Lenti CMV Renilla Control) at a concentration of 1×10⁶ cells/ml in a 24-well tissue culture plate in the presence of polybrene (8 µg/ml). The plates were centrifuged at 800 × g for 90 min at 32°C. The cells were then washed and resuspended in fresh medium for an additional 72 h. Then puromycin and hygromycin were used together for selection. For the drug experiments, cells were subsequently seeded at a density of 4×10⁵ cells/ml and treated for 12 h with the vehicle, ST2825, ibrutinib or a combination of ST2825 and ibrutinib at the indicated concentrations. Luciferase activity was measured in 96-well plates using the Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocol.