Figure 5: Regulation of NF-κB activity by Fbw7 with or without stimuli.

(a) Basal NF-κB activity is lower in HCT116 FBW7 KO compared with WT cells. WT and KO cells were transfected with the kB-luciferase reporter. Relative luciferase activity was measured. Results are shown as means. Error bars indicate s.d. (n=3). (b) p100 overexpression represses kB-luciferase activity in the absence of stimulation. HEK293 or HCT FBW7 WT cells were transfected with the kB-luciferase reporter and empty vector (EV), p100, pSA and NIK as indicated. Results are shown as means. Error bars indicate s.d. (n=3). The p100/EV ratios of fold induction were 29 (s.d.=5) in HEK and 2.9 (s.d.=0.75) in HCT cells. (c) FBW7 WT and KO cells were transfected with the κB-Luciferase reporter with NIK or treated with 5 ng ml⁻¹ TNF-α (24 h). Luciferase activity was analysed and normalized to untreated cells for each cell type. Error bars show s.d. (n=3). (d) U2OS cells were transfected with EV, Fbw7 or Fbw7ΔF as indicated. All cells were also transfected with the cyclin D1 promoter-specific luciferase reporter. Results are shown as means. Error bars indicate s.d. (n=3). (e) Response to the NF-κB pathway stimulation is enhanced in Fbw7ΔF expressing cells. HEK293 cells were transfected with the κB-Luciferase reporter and NIK, Fbw7 and Fbw7ΔF as indicated for 48 h. Luciferase activity was measured. The results were shown as means. Error bars are s.d. (n=3). (f) Protein levels of NF-κB2 targets are higher in FBW7 KO cells. WB analysis of NF-κB2 targets in HCT FBW7 WT and KO cells. Cells were treated with 10 ng ml⁻¹ TNF-α for the indicated times. Statistical significance was tested using ANOVA in (d) and unpaired, two-tailed t-tests elsewhere. *P<0.05; **P<0.01 and ***P<0.001.