Fig. 4.

Identification of 7-oxo metabolites in SLOS plasma and amniotic fluid and demonstration that they modulated Hh signalling by binding to the CRD of vertebrate Smo. LC–MS³ chromatograms of GP-tagged 26H,7−OC (upper panel) and 3βH,7O-CA (lower panel) in SLOS (A) amniotic fluid; and (B) plasma. The identities of the sterols eluting after 26H,7−OC in the amniotic fluid sample and before 26H,7−OC in the plasma sample are unknown. SLOS samples are known to be rich in multiple sterols derived from free radical oxidation of 7-DHC, many of which are yet to be fully characterised [92]. LC–MS³ spectra from SLOS plasma identifying (C) 26H,7−OC (upper panel) and 3βH,7O-CA (lower panel). Spectra of authentic standards can be found in [93]. (D) Levels of Gli1 mRNA (y-axis, mean arbitrary units ± SD, n = 3) were used as a metric for Hh signalling activity in NIH/3T3 cells after stimulation (7.5 h) with oxysterol (10 μM). With the exception of 3β,7β-diHCA the oxysterol agonists induced Gli1 mRNA at 1 μM. (E) Immunoblots showing the amount of zSmo CRD captured on 20S−HC beads in the presence of the indicated competitors.