Figure 6. 7-DHC supports Ret signaling.

(A) MG87 cells were treated or not with MβCD for 30 min to deplete cholesterol (stripping), followed by 30 min replenishment step with MβCD alone, loaded with 7-DHC or with cholesterol, or with 7-DHC in the presence of the Dhcr7 inhibitor BM15766. Cells were then stimulated with GDNF for 10 minutes. Total lysates were resolved by SDS-PAGE and probed with the indicated antibodies. (B) Representative extracted ion chromatograms and MS spectrum for 7-DHC and Cholesterol in samples from cells replenished with MβCD:7-DHC complexes. Chromatograms shown retention times of the compounds and the MS peak used for quantification. The ions selected for quantification was m/z (M + H) + (−H2O): 367.3360 for 7-DHC and m/z (M + H) + (−H2O): 369.3519 for cholesterol. (C) Relative abundances of 7-DHC and cholesterol measured by means of LC-QTOF-MS in two independent experiments. Numbers indicate the ratio of the indicated metabolite (7-DHC or cholesterol) with respect to an internal standard (¹³C-cholesterol). N.d.: not detected.