Figure 5. The effect of the Shh pathway on Snail expression.
A, The effect of the Shh pathway inhibitors on Snail expression. KAT-18 cells were treated with cyclopamine (CP) (5 μm), HhAntag (10 μm), or GANT61 (10 μm) for 72 hours. The cells were harvested and analyzed for Snail and Actin expression by Western blot with their specific antibodies. B, Regulation of Snail by the Shh pathway. KAT-18 cells transfected with the indicated genes were analyzed for Snail expression by Western blot. C, Snail knockdown. KAT-18 cells were transfected with the indicated amounts of Snail or scrambled siRNA as a control. SW1736 cells were transfected with control (Ctr) or Snail siRNA (2.5 nmol each). After incubation for 48 hours, the cells were harvested and analyzed for Snail and actin expression by Western blot. The experiments were repeated twice with similar results. D and E, The effect of Snail knockdown on KAT-18 (D) and SW1736 (E) CSC self-renewal. Cells seeded in 35-mm dishes were transfected with a scrambled control siRNA or Snail siRNA (2.5 nmol each). After incubation for 72 hours, the cells were analyzed for ALDHHigh thyroid CSCs by ALDEFLUOR assay.