Hh signaling pathway is involved in the expression of p21, cyclin D1 and regulation of cell migration. AGS, BGC823 and SGC7901 gastric cancer cells were treated with cyclopamine (10 μM), a steroidal alkaloid that interacts directly with Smo to inhibit Hh signaling, or DMSO control for 24 h. (A) The expression levels of p21 and cyclin D1 were determined by western blot in AGS, BGC823 and SGC7901 cells. (B) The expression level of ZIC1 mRNA was examined through conventional RT-PCR. (C) Cell migration was assessed by modified Boyden transwell chambers assays. Cells that migrated to the bottom of the membrane were stained with DAPI. (D) The mean number of visible migratory cells (mean ± S.D) was calculated in five random high power fields (×400). The asterisk indicates statistical significance (***p < 0.001).