Purmorphamine (Shh Signaling Antagonist VI), which directly binds and activates Smoothened, blocks BODIPY-cyclopamine binding to Smo. It also is an inducer of osteoblast differentiation.

Smo:1.5 μM

Binding assay: Smo binding assays are conducted with BODIPY-cyclopamine and Smo-overexpressing cells as previously described4,5, using CMV promoter-based, SV40 origin-containing expression constructs for Smo-Myc3, the deletion mutant Smo?CRD (deletion of amino acids 68 to 182), and Smo?CT (deletion of amino acids 556 to 793). HEK 293T cells are grown on poly-D-lysine-treated glass coverslips in 12-well plates until 70% confluency and then transfected with the appropriate expression construct (0.5 ?g/well) using FuGene 6 according the manufacturer's protocols. Two days after transfection, the HEK 293T cells are incubated with DMEM containing 0.5% bovine calf serum, 5 nM BODIPY-cyclopamine, and varying concentrations of Purmorphamine (0, 1.5, or 5 ?M) (1 mL/well) for 1 h at 37 ℃. The Smo-overexpressing cells are then washed with 1 × PBS buffer (1 mL/well), mounted with DAPI-containing medium, and visualized using a Leica DM4500B fluorescence microscope. For binding assays using fixed cells, the Smo-overexpressing HEK 293T cells are fixed with 3% paraformaldehyde in 1 × PBS buffer for 10 min at room temperature (1 mL/well), treated with 1 × PBS containing 10 mM glycine and 0.2% sodium azide for 5 min (1 mL/well), washed with 1 × PBS buffer (1 mL/well), and treated with the Purmorphamine-containing media described above for 4 h at room temperature.

C3H10T1/2 cells are expanded in T175 flasks; cells at 13th passage are detached by trypsin/EDTA and diluted in the growth media. The resulting cell suspension is then plated into black clear bottom 384-well plates with 2500 cells/well in 100 μL growth medium using a Multi-dropTM liquid delivery system. After overnight incubation, cells attached to the bottom of the wells. A stock solution of each Purmorphamine in DMSO (500 nL) is delivered into corresponding well using a Mini TrakTM multiposition dispenser system to make a final concentration of 5 μM of Purmorphamine. Cells are then incubated at 37 ℃ with 5% CO2 in air atmosphere. After 4 days, the medium is removed and 10 μL of passive lysis buffer is added into each well. After 5 min, 10 μL of alkaline phosphatase substrate solution is added to each well. After incubating 15 min at room temperature, the plates are read on an Acquest high-throughput plate reader following the manufacturer's protocol.(Only for Reference)