Figure 4. Gli1 overexpression stimulates thyroid CSC self-renewal.
A, Luciferase reporter assay. pcDNA3.1- or pcDNA/Gli1-transfected cells were transfected with the pGL3/Basic empty vector or the same vector encoding an 8XGli1-Luc reporter gene. Luciferase activity was analyzed 48 hours later. **, The P value in comparison with pcDNA3.1 transfected control was < .01. B, ALDEFLUOR assay. Single cell suspensions of KAT-18 cells transfected with pcDNA/3.1 or pcDNA/Gli1 were analyzed for ALDHHigh cells by ALDEFLUOR. The experiments were repeated twice with similar results. C, Thyrosphere formation. Single cell suspension of pcDNA3.1 and pcDNA/Gli1-transfected KAT-18 cells were resuspended in serum-free DMEM/Ham's F-12 (1:1) medium containing B-27, EGF, and bFGF (20 ng/mL each) and seeded with 200 cells per well in a 96-well ultralow-attachment plate. Thyrospheres were counted and presented as the mean ± SD from 48 wells in a representative experiment. **, The P value in comparison with pcDNA3.1 transfected control was < .01.