Figure 4. GLI1^3A has higher protein stability and transcriptional activity.

The GLI1^3A mutant has increased stability and is resistant to AMPK-mediated suppression of Gli1 transcriptional activity. (A) Lysates of HEK-293 cells transfected with Flag–GLI1^WT, Flag–GLI1^3A, or Flag–GLI1^3E, were harvested at different times after treatment with cycloheximide (CHX, 1 μg ml⁻¹), and analyzed by immunoblot. GLI1^3E has the two serines and one threonine that are normally phosphorylated by AMPK changed into glutamates to mimic phosphorylated GLI1. (B) HEK-293 cells were co-transfected with Gli1-luciferase reporter, Gli1^WT and AMPK for 36 hours. The two amounts of AMPK-expressing plasmid used were 1 and 3ug. Cell lysates were analyzed using a luciferase assay to measure GLI1 transcriptional induction of the introduced Gli1-luciferase target gene. Representative results from three experiments (n = 3), each conducted in duplicate, are shown with standard deviations. (C) HEK-293 cells were co-transfected with Gli1-luciferase reporter, GLI1^3A, and AMPK and analyzed, as in (B). (D) NIH3T3 cells were transfected with vector control, GLI1^WT, or GLI1^3A for 36 hours, then treated with 2DG (25mM) for 4 hours. RT-PCR was used to measure (D) Gli1 mRNA and (E) Ptch1 mRNA. The experiment was repeated three times. (“ ** ” p<0.01, “ *** ” p<0.001) (F) NIH3T3 vector, GLI1^WT and GLI1^3A producing stable cell lines were treated with AICAR (0.75mM) and A769662 (150 M) for 4 hours. RT-PCR was used to measure (F) Gli1 mRNA and (G) Ptch1 mRNA. The experiment was repeated three times.