Figure 8: Influence of selected compounds on Hedgehog signalling.

(a) Chemical structures of 11b (structure for the major diastereomer is shown), 15g and 16c. (b) Influence of 11b, 15g and 16c on purmorphamine-induced osteogenesis in C3H10T1/2 cells as determined by the activity of alkaline phosphatase. C3H/10T1/2 cells were treated for 96 h with 1.5 μM purmorphamine and different concentrations of the compounds or DMSO as control. Activity of alkaline phosphatase was determined using a luminescent readout. Nonlinear regression was performed using a four parameter fit. Data are mean values (n=3)±s.d. and were normalized to purmorphamine-treated cells. (c) Influence of 11b, 15g and 16c on the relative expression of Ptch-1. NIH/3T3 cells were incubated with 2 μM purmorphamine and different concentrations of the compounds or DMSO as control for 48 h. Following cDNA preparation, the relative expression levels of Ptch-1 and Gapdh were determined by means of quantitative PCR. Data are mean values (n=3)±s.d. and were normalized to purmorphamine-treated cells (*P<0.05, **P<0.01 and ***P<0.001). (d) Influence of 11b, 15g and 16c on Gli-mediated reporter gene expression. Shh-LIGHT2 cells were treated with 4 μM purmorphamine and different concentrations of the compounds or DMSO as control for 48 h. Luciferase activity was determined as a measure of Hedgehog pathway activity. Data are mean values (n=3)±s.d. and normalized to cells treated with purmorphamine. (e) Influence of the compounds on the binding of BODIPY-cyclopamine to Smo. HEK293T cells were transfected with a Smo-expression construct. Two days later, cells were treated with the compounds or DMSO as control in the presence of 5 nM BODIPY-cyclopamine for 5 h. Cells were then subjected to flow cytometric analysis to detect Smo-bound BODIPY-cyclopamine. The graph shows the median BODIPY-cyclopamine fluorescence intensity on treatment with the compounds. Data are mean values (n=3)±s.d.