(a) Structure of cholesterol, which carries a hydroxyl group at the 3 position, with colored circles marking the positions of the second hydroxyl group on the oxysterols tested. (b) The mean (± SEM) Hh luciferase reporter activity from triplicate wells was measured at various concentration of the indicated oxysterols and plotted as the fold-change compared to activity measured from control cells treated with solvent (ethanol). The black dotted line shows the reporter activity produced by a saturating concentration (100 nM) of the agonist SAG. A non-linear curve fit to the 20(S)-OHC response (blue line) yields an EC50 of ~3 μM. (c) Confocal images of ciliated NIH 3T3 cells treated with solvent (control) or the indicated oxysterol (10 μM). The cilia marker acetylated tubulin (red) and Smoothened (green) were both detected by immunofluorescence; nuclei (blue) were stained with DAPI. The inset is a zoomed image of a single cilium where the green channel is shifted relative to the red channel. Scale bars: 5 μm. (d) Each dot represents Smo fluorescence at a single cilium. Red bars depict the mean and 95% confidence interval (n~35).

(a) Structures of nat-20(S)-OHC (1), nat-20(R)-OHC (2) and ent-20(S)-OHC (3). Colored dots denote chiral carbon centers. ent-20(S)-OHC is the mirror image of nat-20(S)-OHC, such that the stereochemistry at each of the eight chiral centers is reversed. nat-20(R)-OHC is a diastereomer of nat-20(S)-OHC, with only the stereochemistry at the C-20 position reversed. (b) Mean (± SEM) Gli reporter activity, expressed as fold-change relative to a control treated with ethanol alone in NIH 3T3 cells treated with oxysterols. Results with this Gli reporter construct were confirmed by measuring increases in endogenous Gli1 protein by immunoblotting (c, Supplementary Fig. 10) and Gli1 RNA (e) by q-RT-PCR after treatment with oxysterols (10 μM). (d) nat- and ent-20(S)-OHC produce identical levels of fluorescence dequenching when added to carboxyfluorescein-loaded unilamellar vesicles. Controls were run in parallel with each oxysterol. (f) Quantification of ciliary Smo fluorescence in NIH 3T3 cells treated with solvent control or 10 μM of the indicated oxysterols (n~35). Each dot represents one cilium and the red bar represents the mean bracketed by the 95% confidence interval. (g) Hh reporter activation was measured at various concentrations of the indicated isomers of 20(S)-OHC and 25-OHC.

(a) nat-20(S)-OHC could activate the Hh signaling by directly inhibiting Ptc1, directly activating Smo or by modulating an intermediate step. (b) Cyclopamine inhibition curves in cells treated with nat-20(S)-OHC (8 μM) or Shh-containing conditioned media (Shh-CM) used at a ¼ dilution (1/4 Shh). The main graph shows Gli luciferase reporter activity, normalized such that the reporter activities with no cyclopamine and maximal cyclopamine are set to 100% and 0% respectively. Inset shows the ratio of Gli to Renilla luciferase activity without normalization (AU: arbitrary units). (c) The IC50 of cyclopamine is not affected by the dose of nat-20(S)-OHC used to activate Hh signaling. (d) Cyclopamine reduces the maximum level of Hh pathway activity driven by saturating concentrations of nat-20(S)-OHC. The IC50 of SANT-1 (e) and SANT-2 (f) increases with increasing doses of nat-20(S)-OHC, but the IC50 of itraconazole is unaffected (g).

(a) Dose response curves of nat-20(S)-OHC in the presence of low concentrations of SAG. The EC50 of nat-20(S)-OHC shifts from 3 μM without SAG to 0.24 μM with 0.3 nM SAG. (b) The reciprocal experiment shows that the EC50 of SAG drops from 2 nM with no nat-20(S)-OHC to 0.4 nM with 0.5 μM nat-20(S)-OHC. The dotted line denotes reporter activity produced by 8μM nat-20(S)-OHC. A combination of low concentrations of SAG and nat-20(S)-OHC can fully activate Hh signaling as measured by the induction of endogenous Gli1 protein levels (c, Supplementary Fig. 10) and the accumulation of Smo in cilia (d). The low concentrations of each agonist used have minimal effects when added individually to cells (a–d). (f) Low doses of Shh-CM (1/64 and 1/128 dilutions) do change the EC50 of nat-20(S)-OHC. (f) The observed Hh reporter activity from (a) and (e) and the predicted reporter activity based on a purely additive Bliss model is plotted at various doses of nat-20(S)-OHC in combination with either low Shh or low SAG. (g) Low doses of purmorphamine can reduce the EC50 of nat-20(S)-OHC. (h) Bliss independence analysis of the data in (g) suggests synergy between nat-20(S)-OHC and purmorphamine. Error bars in all graphs represent SEM.

Chemical structures of nat-20(S)-yne (4), containing a terminal alkyne compatible with click chemistry, a nat-20(S)-yne derivative (5) which is 4 coupled to a PEG linker, and 5 immobilized on magnetic beads. (b) A Gli-luciferase reporter assay demonstrates that nat-20(S)-yne (EC50 ~390 nM) is 8-fold more potent at activation of Hh signaling compared to nat-20(S)-OHC. (c) An immunoblot, depicted at two exposures, showing the amount of YFP-Smo precipitated by nat-20(S)-yne-beads or control beads from membrane extracts made from either smo−/−:YFP-Smo cells or smo−/− cells. The complete immunoblot is included in Supplementary Fig. 10. (d) Control beads or nat-20(S)-yne beads were incubated with membrane extracts made from 293T cells expressing YFP-Smo, a GFP-tagged somatostatin receptor (SSTR3-GFP) or a GFP-tagged serotonin receptor (HTR6-GFP). The amount of YFP/GFP-tagged receptor precipitated in each case was measured from the immunoblot shown in Supplementary Fig. 8c and plotted. (e) Free nat-20(S)-yne can inhibit the binding of YFP-Smo to nat-20(S)-yne beads (IC50=1.3μM). Error bars represent the SEM from three independent experiments. (f) nat-20(S)-yne beads incubated with YFP-Smo containing extracts were washed and subjected to ligand-elution with the indicated sterols (500μM each). The amount of YFP-Smo eluted in each case was measured by immunoblotting (Supplementary Figs. 8d and 10).

The key postulate of this model is the presence of two distinct binding sites on Smo, one for SAG and one for nat-20(S)-OHC, that show a positive allosteric interaction, denoted by the zig-zag line. Cyclopamine binds to a site that overlaps with SAG and thus is an orthosteric competitive antagonist of SAG. However, the effect of cyclopamine is “agonist-specific” because it acts as a non-competitive antagonist of nat-20(S)-OHC. When cyclopamine binds to Smo, nat-20(S)-OHC cannot activate the protein even at saturating concentrations. The active conformation of Smo, one that is capable of transducing the Hh signal, is colored green and the inactive conformation is colored gray.