NS-398-induces NF-κB DNA-binding activity but not transcriptional activity in HT-29 cells. HT-29 cells were transfected with either the firefly luciferase reporter vector pNF-κB-TA-luc or with the control vector pTA-luc lacking NF-κB binding sites. All transfections also included the constitutively expressed renilla luciferase vector pRL-SV40 and hence firefly luciferase readings were normalised to renilla luciferase activity in order to control for transfection efficiency. (A) NS-398 does not increase NF-κB-dependent transcriptional activity in HT-29 cells. Cells transfected with pTA-luc or pNF-κB-TA-luc were then treated with 0 (vehicle) or 75 μ M NS-398 for 72 h. After this time, PLB lysates, were prepared and assayed for firefly and renilla luciferase activities. Luminescence data are expressed as mean firefly:renilla luciferase ratios±s.d. of three independent experiments and within each experiment samples were assayed in triplicate. (B) and (C) TNF-α activates NF-κB-dependent transcriptional activity at doses inducing a similar NF-κB DNA-binding activity to 75 μ M NS-398 in HT-29 cells. (B) Cells transfected with pTA-luc or pNF-κB-TA-luc were treated with a range of doses of TNF-α for 24 h. After this time lysates were prepared and assayed for firefly and renilla luciferase activities. Luminescence data are expressed as mean firefly : renilla luciferase ratios±s.d. from one representative of three independent experiments carried out in triplicate (means were not taken in this case due to different activities between batches of TNF-α necessitating the use of differing doses of TNF-α between experiments). (C) Nuclear protein extracts (from pNF-κB-TA-luc-transfected cells) were prepared using parallel flasks to those from (A) and (B) and analysed by EMSA for each of the three independent experiments.