Fig. 5: Biological investigation of PNP A23 for Hh signalling inhibition.

a, Selected compounds with structural variation to the most active compound (A23) in an osteoblast differentiation assay. Cell viability was assessed using a CellTiter-Glo Luminescent Cell Viability Assay and treating C3H10T1/2 cells with compound (30 µM) in the absence of purmorphamine for 96 h. The viability of cells treated with DMSO was set to 100%. IC₅₀, half-maximal inhibitory concentration. b, C3H10T1/2 cells were treated for 96 h with 1.5 µM purmorphamine, DMSO as a control or compound A23. The activity of alkaline phosphatase was measured to determine Hh pathway activity. Values for cells treated with purmorphamine and DMSO were set to 100%. The data are the mean values ± s.d. of three biological replicates (n = 3). c, Alpl gene expression; C3H10T1/2 cells were incubated for 96 h with 1.5 µM purmorphamine and DMSO, 1 µM vismodegib (vismo) or 1, 5 or 10 µM of A23 before RT–qPCR. Data are mean values ± s.d. of three biological replicates (n = 3). The P values relative to cells treated with DMSO and purmorphamine are <0.0001 for DMSO-treated, <0.0001 for vismo (1 µM)-treated, 0.0014 for A23 (1 µM)-treated, 0.0005 for A23 (5 µM)-treated and 0.0006 for A23 (10 µM)-treated cells. d,e, Expression of the Hh target gene Gli1 (d) and Ptch1 (e). C3H10T1/2 cells were incubated with 1.5 µM of purmorphamine and DMSO, 1 µM vismo or A23 (1 µM, 5 µM or 10 µM) for 96 h before RT–qPCR. Data are mean values ± s.d. of three biological replicates (n = 3). For Hh target gene Gli1, the P values relative to cells treated with DMSO and purmorphamine are 0.0001 for DMSO-treated, 0.0003 for vismo (1 µM)-treated, 0.0477 for A23 (1 µM)-treated, 0.0052 for A23 (5 µM)-treated and 0.0203 for A23 (10 µM)-treated cells. For Hh target gene Ptch1, the P values relative to cells treated with DMSO and purmorphamine are 0.0010 for DMSO-treated, 0.0024 for vismo (1 µM)-treated, 0.4559 for A23 (1 µM)-treated, 0.0111 for A23 (5 µM)-treated and 0.0142 for A23 (10 µM)-treated cells. f, SMO binding assay. HEK293T cells were transfected with a SMO-expressing plasmid. After 48 h, the cells were fixed and incubated with BODIPY–cyclopamine (green, 5 nM) and treated with either DMSO, vismo or A23 for 4 h. The nuclei were visualized by staining the cells with 4,6-diamidino-2-phenylindole (DAPI) (blue). The images are representative of three biological replicates (n = 3). Scale bar, 30 µm. For c–e, statistical analyses were performed relative to DMSO/purmorphamine by employing unpaired two-tailed t-tests with Welch’s correction (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant).