Cyclopamine

All animal care and treatments were done as approved by Animal Use and Care Committee of Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH). Adult Xenopus laevis were purchased from NASCO. Tadpoles were obtained by breeding wild type frogs, and were staged according to [64 ].
Stage 58 Xenopus laevis tadpoles of similar sizes were treated with cyclopamine (Enzo life sciences) (2.5 μM), tomatidine (Enzo life sciences) (2.5 μM), or vehicle (100% ethanol, final concentration in rearing water: 0.1%), respectively, at room temperature. The rearing water was changed every two days.

Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich (St Louis, MO). SAG, GSA-10, GDC-0449, MRT-92 and LDE225 were synthesized as described16 (link)23 (link)37 (link). Purmorphamine and recombinant Shh (C25II N-Term, RecShh) were purchased from Calbiochem and R&D Systems, respectively. Cyclopamine was purchased from Enzo Life Sciences and KAAD-Cyclopamine was from Santa Cruz Biotechnologies. SANT-1 was obtained from TOCRIS. SAG and Cyclopamine were dissolved in ethanol, while the other compounds were dissolved in DMSO at a concentration of 10 mM, except GSA-10, which was at 2.5 mM. Unless specified, the following concentrations of these compounds were used: SAG (200 nM), GSA-10 (10 μM), recShh (0.5 μg/mL), KAAD-Cyclopamine and Cyclopamine (10 μM), SANT-1 (1 μM), LDE225 (3 μM), MRT-92 (1 μM) and GDC-0449 (3 μM).

Smoothened agonist (SAG) was purchased from Calbiochem (San Diego, CA; 566660). Receptor activator of NF-kappa-B ligand (RANKL) was purchased from Pepro Tech (Rocky Hill, NJ; 184-01791). Sonic hedgehog (Shh) was purchased from R&D Systems (Minneapolis, MN; 1845-SH). Cyclopamine was purchased from Enzo Life Sciences (Farmingdale, NY; BML-GR3334). Plasmids expressing human GLI1 were constructed as previously described [12] (link). The adenoviral vector expressing human GLI1-Biotin-3xFLAG-IRES-dsRed was constructed using the pAd/PL-DEST vector and ViraPower Adnoviral Expression System (Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. In brief, human GLI1 cDNA carrying Biotin-3xFLAG tag [16] (link) was initially cloned into pCID vectors [17] (link); GLI1-Biotin-3xFLAG-IRES-dsRed was then transferred into the pENTR1A vector in conjunction with the CAGGS promoter and subjected to adenoviral vector construction using the Gateway system. The pAd/PL-DEST expressing the CAGGS promoter-driven GLI1-Biotin-3xFLAG-IRES-dsRed was linearized with Pac I and transfected into 293A cells. After amplification, the virus was stored at −80°C. The viral titer was determined by an end-point titer assay using 293A cells.