Figure 3. Shh or Gli1 knockdown suppresses thyroid CSC self-renewal.
A, Western blot analysis of Gli1 and Shh expression. KAT-18 cells stably transfected with Ctr-miRNA, Shh-miRNA (constructs that target Shh miRNA at the nucleotide 658 or 1201), or Gli1-miRNA (constructs that target Gli1 miRNA at the nucleotide 760 or 1519) were analyzed for Shh, Gli1, and actin expression by Western blot. B, KAT-18 cells stably transfected with Ctr-miRNA, Shh-miRNA 658, or Gli-miRNA 1519 were analyzed for ALDHHigh cells by ALDEFLUOER. The experiments were repeated at least twice with similar results. C, Thyrosphere formation. Single cell suspension of Ctr-miRNA, Shh-miRNA, or Gli1-miRNA-transfected KAT-18 cells was resuspended in serum-free DMEM/Ham's F-12 (1:1) medium containing B-27, EGF, and bFGF 20 ng/mL each and seeded at 2 × 103 cells per well in six-well ultralow-attachment plates. A representative of thyrospheres from these transfected cell lines was photographed 2 weeks after seeding. Thyrospheres were counted and presented as the mean ± SD from one of two experiments in triplicate. **, P < .01.