GDC-0449 was purchased from Selleck Chemicals (Texas, USA). SHH signaling agonist SAG was obtained from Enzo Life Sciences (NewYork, USA) and recombinant mouse sonic hedgehog N-terminus was obtained from R&D Systems selleck chemicals (Minnesota, USA). Agonist and Antagonist Treatment Agonists or antagonists were added when irradiated HT29 or Panc1 cells were seeded into 24 well plates as feeders. The following concentrations were used: cyclopamine for HT29 cells (2 ��M and 5 ��M), cyclopamine for Panc1 cells (0.5 ��M, 1 ��M, 2 ��M and 5 ��M), GDC-0449 for HT29 cells (0.2 ��M, 0.5 ��M, 1 ��M and 2 ��M), GDC-0449 for Panc1 cells (0.5 ��M, 1 ��M and 2 ��M), Gant61 for HT29 cells (1 ��M, 2 ��M and 5 ��M), Gant61 for Panc1 cells (2 ��M, 5 ��M, 10 ��M and 20 ��M), recombinant mouse sonic hedgehog N-terminus for HT29 and Panc1 cells (600 ng/ml), SAG for HT29 cells (5 nM, 10 nM and 100 nM), SAG for Panc1 cells (3 nM, 5 nM, 10 nM and 100 nM).

To confirm the effect of agonists on live tumor cells, agonists were also added into wells containing HT29 or Panc1 reporter cells alone. The concentrations were used as: SAG for Panc1 and HT29 cells (5 nM, 10 nM and 100 nM), recombinant mouse sonic hedgehog N-terminus peptide for HT29 and Panc1 cells (600 ng/ml). Medium was changed every forty-eight hours and replaced with fresh medium containing same concentration of agonists or antagonists. The growth of reporter cells was monitored 14 days later by imaging. ShRNA Knockdown The lentiviral plasmids encoding shRNA against Gli1 gene (HSH007701-HIVmU6) and scramble control (CSHCTR001-HIVmU6) were purchased from Genecopoeia (Maryland, USA).

The sequence for shRNA against Gli1 is 5��-acgccatgttcaactcgat-3��. Lentiviruses encoding Gli1 shRNA and scramble control were produced as described above. HT29 and Panc1 cells were seeded in six well plates and subsequently infected. The cells were then treated with puromycin to select for those stably expressing shRNA against Gli1 and scramble control RNA. Silencing efficiency was confirmed using Western blot for Gli1 protein. Western Blotting Cells were washed twice with PBS and lysed using 120�C200 ��l standard RIPA buffer containing protein inhibitors (Beyotime, Jiangsu, China). Protein concentration GSK-3 of each sample was quantified and 40�C60 ��g protein per sample was used for Western blot analysis. In general, 40�C60 ��g protein in loading buffer was heated to 100��C for 10 minutes and then separated in a SDS-polyacrylamide gel by electrophoresis, and transferred to a PVDF membrane (Bio-Rad, California, USA). The membranes were incubated with primary antibodies overnight at 4��C and then with secondary antibodies for 2 hours at room temperature. ECL Plus (Roche, Basel, Switzerland) was used to visualize the signals on the membrane.