Figure 4. Mapping of itraconazole action within the Hh pathway.

(A) Itraconazole inhibited constitutive Hh pathway activity in Ptch−/− cells, measured as expression of β-galactosidase activity from the Ptch-lacZ locus (Goodrich et al., 1997; Taipale et al., 2000). The levels of β-galactosidase activity from cells treated with KAAD-cyclopamine at 40 and 200 nM (2× and 10× IC₅₀) are shown as purple and red lines, respectively. (B) Shh-Light SmoA1 cells, stably expressing a constitutively active Smo mutant, were resistant to Hh pathway inhibition by itraconazole. Cyclopamine and forskolin (orange and blue lines, respectively) were used as negative and positive controls for the inhibition of SmoA1. (C) Itraconazole and KAAD-cyclopamine both inhibited constitutive Hh pathway activation induced by transient overexpression of Smo in NIH-3T3 cells. (D) Itraconazole inhibited Hh pathway activity induced by the combined oxysterols, 20(S)-and 22(S)-hydroxycholesterol (5 µM each). All signaling assays were performed with Shh-Light2 cells in 0.5% serum media and data are shown as the mean of triplicates ± s.d. (E) Representative immunofluorescent images of NIHT-3T3 cells. Smo accumulated in primary cilia upon stimulation with ShhN (arrowheads). This accumulation was blocked by treatment with itraconazole. Insets show enlarged views of primary cilia in the dashed box, with Smo and acetylated tubulin channels offset. Arrows show primary cilia with no Smo accumulation. As seen in the histogram, the accumulation of Smo in ~90% of primary cilia of cells stimulated with ShhN, is reduced to ~5% in the additional presence of itraconazole. The number of primary cilia containing Smo over the total number of primary cilia are shown above each bar. Data are mean of 10 images ± s.d. The scale bar represents 5 µm.