Fig. 4.

ATO attenuates the activity of the activator and repressor forms of GLI proteins. (A) GLI2^−/−;GLI3^−/− MEFs were transfected with a plasmid expressing a GLI luciferase reporter gene with or without GLI1 or GLI2 expression plasmids and then treated with the indicated doses of ATO. Luciferase activity relative to total protein level was measured after 48 hours of treatment. The activity of a vehicle-treated and mock-transfected sample (left) was set as 1 for comparison. Error bars represent the S.E.M. (n = 3). *P < 0.05. (B) GLI2^−/−;GLI3^−/− or WT MEFs were transfected with a plasmid expressing a GLI luciferase reporter gene and then treated with the indicated doses of ATO. Luciferase activity relative to total protein level was then determined. The activity of a vehicle-treated sample (left, WT MEFs) was set as 1 for comparison. Error bars represent the S.E.M. (n = 3). *P < 0.05. (C) GLI3^−/− MEFs were transfected with increasing amounts of a GLI3-R expressing plasmid and then treated with vehicle or 3 µM ATO. PTCH1 expression relative to that of GAPDH was measured as a readout of GLI activity. PTCH1 expression relative to that of GAPDH in WT MEFs was also determined as a control. Expression in a vehicle-treated, mock-transfected sample (left) was set as 1 for comparison. Error bars represent the S.E.M. (n = 3). *P < 0.05. (D) GLI3^−/− MEFs were transfected with the indicated amounts of GLI3-R expression plasmids and then treated with vehicle or 3 µM ATO. GLI3-R (a long and short exposure) or GAPDH levels were revealed by immunoblotting. A representative result is shown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.