Purmorphamine

Purmorphamine activates the Hedgehog pathway by directly binds and activates Smoothened with IC50 of ~ 1.5 μM in compete with cyclopamine, a Smo antagonist. ^[1]

Purmorphamine is a potent inducer of osteogenesis in multipotent C3H10T1/2 cells. The EC50 (based on ALP expression) for Purmorphamine is 1 μM in C3H10T1/2 cells. Purmorphamine (1 μM) and BMP-4 (100 ng/mL) together increase ALP activity more than 90-fold in 3T3-L1 cells. ^[2]

In contrast to BMP-4, Purmorphamine induces osteogenesis by activating Hedgehog signaling in multipotent mesenchymal progenitor cells. ^[3]

Purmorphamine up-regulates ALP expression in human mesenchymal stem cell-based constructs on rats. ^[4]

• 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

• Cell lines

  C3H10T1/2 cell

• Concentrations

  0.5-10 μM

• Incubation Time

  4 days

• Method

  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-drop^TM 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 Trak^TM multiposition dispenser system to make a final concentration of 5μM 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.

• Animal Models

  Male C57BL/6J mouse pups

• Dosages

  10 mg/kg

• Administration

  i.p.