Figure 10. Analysis of GLI1-driven transcription by GLI1 variants.
(A) COS-1 cells were transfected with a luciferase reporter vector for GLI-driven transcription, TK-βgal (a control for transfection efficiency) and either pCMV6-FL-GLI1, pCMV6-FL-GLI1AAA, or pCMV6-FL-GLI1EEE, as indicated. Cells were collected 24 h later and assayed for reporter activity. Luciferase activity was first normalized to β-galactosidase and then normalized again so that wild-type activity equals 100 units. (B) NIH3T3 cells stably expressing full-length human GLI1 variants from piggyBac transposons were serum starved for 24 h and then treated with Shh-N conditioned media or control conditioned media. Expression of the endogenous mouse Gli1 locus was analyzed by real-time quantitative RT-PCR. Fold change in mouse Gli1 mRNA expression was measured using ΔΔCt analysis. mApple (expressed from the same transposon) served as an internal control. The data were further normalized so that uninduced wild type equals 1 unit. *P < 0.05, **P < 0.01, ***P < 0.001.