Figure 4

Regulation of GLI induced transcriptional activity in transfected HEK293 cells. HEK293 cells were transfected with a GLI inducible luciferase reporter construct together with a GLI1 or GLI2 expression construct. The cells were also transfected with a β-galactosidase construct that served to correct for transfection efficiency and cell density. The effects of FU and SUFU were tested by cotransfecting FU and SUFU expression constructs and compared to effects with empty vectors. Panel A shows typical examples of SUFU effects on GLI1 and GLI2. Panel B shows typical examples of the effects of different FU constructs on GLI1 and GLI2; experiments that were performed in parallel are shown, to illustrate the different impact on the GLI proteins. L-FU is shown with squares, 2HFU with triangles and S-FU with circles. Panel C shows the impact of FU constructs (400 ng) on GLI1 and GLI2 as summarized by the results of at least 4 different experiments. Panel D shows the impact of FU constructs (400 ng) on SUFU inhibited GLI1 and GLI2 as summarized by the results of 3 different experiments. In these experiments the GLI induced transcriptional activity was inhibited to 20–40 % of the non-inhibited level by the addition of SUFU (i.e. 2½ to 5 fold inhibition). Relative activity (panel A) is given as compared to activity in mock transfected cells and normalized activity (panel B-D) is given relative to activity in cells transfected with GLI and GLI+SUFU set to 1.