FIGURE 8.

Prolonged treatment with PMA stimulates canonical and non-canonical NF-κB activity. A, transcription factor binding sites in the −163 bp region of the cyclin D1 promoter. The position of the indicated transcription factor binding sites in the cyclin D1 promoter is given as the number of bp from the transcriptional start site. Note that there are two binding sites for SP1 and Egr1. This figure is modified from Ref. 70. B, time course for PMA induction of NF-κB activity in IEC-18 cells. Cells transfected with an NF-κB reporter construct containing three NF-κB sites driving expression of firefly luciferase were treated with PMA for various times as indicated. NF-κB activity at each time point was measured by the Dual-Luciferase activity assay and normalized to that in cells treated with vehicle (EtOH) for the equivalent time. *, statistically different from vehicle (EtOH)-treated control cells (p < 0.05). C, effect of dominant active IκBα on PMA-induced NF-κB activity. IEC-18 (i) or DLD1 (ii) cells were transfected with the NF-κB reporter and treated with PMA or vehicle for 8 h. Where indicated, cells were also transfected with a vector expressing dominant active IκB (D.A. IκB) or empty vector (Con). pRL-TK was included in all transfections to control for transfection efficiency; promoter activities in the different transfection conditions were normalized using pRL-TK activity in vehicle-treated cells and are expressed relative to control NF-κB reporter/empty vector-transfected cells. *, statistically different from vehicle (EtOH)-treated control cells (p < 0.05). Data are representative of three independent experiments. Error bars, S.E.