Supplemental Material

This article contains the following supporting material:

• Figure 1 - Suppression of 25-OH activation of SM synthesis in two clones of CHO-K1 cells stably transfected with the shOSBP vector. (A) shNT, shOSBP1 and shOSBP2 cells were treated with increasing concentrations of 25OH for 4 h and pulse-labeled with [³H] serine for the final 2 h. SM was extracted from cells, resolved by thin-layer chromatography and detected by fluorography. (B) Quantification of results from (A) for [³H] serine incorporation into SM in shNT controls (solid squares) and the two knockdown cell lines (solid triangles). Values are mean and range of two independent experiments.

• Figure 2 - Depletion of OSBP by transient transfection of siRNA prevents 25OH activation of SM synthesis. (A) CHO-K1 cells were transiently transfected with non-targeting (siNT) and OSBP- and/or CERT-specific siRNAs and SM synthesis was quantified by [³H] serine incorporation as described in the legend of Figure 2. Results are the mean and standard deviation of a single experiment performed in triplicate that was repeated twice with similar results. (B) Immunoblot analysis of OSBP protein expression in CHO-K1 cells transiently transfected with an OSBP-specific siRNA.

• Figure 3 - OSBP and CERT are not involved in regulation of cholesterol synthesis by 25OH. shNT and shOSBP cells were transfected with non-targeting (NT) or CERT-specific siRNA. Cells were treated with 25OH (6 μM, dark grey bars) or solvent alone (light grey bars) and [¹⁴C] acetate incorporation into cholesterol and fatty acids (FA) was quantified as described in Materials and Methods. Results are the mean and standard error for 3 experiments.

• Figure 4 - Organization of the Golgi apparatus in OSBP- and CERT-depleted CHO-K1 cells. shNT and shOSBP cells were transiently transfected with siNT or siCERT, treated with 25OH (6 μM) or no addition (NA) for 2 h, and immunostained with a polyclonal antibody against giantin followed by an AlexaFluor488 conjugated goat anti-rabbit secondary antibody.

• Figure 5 - VAP does not translocate to the Golgi in response to 25-OH. shNT cells were treated with 25-OH (6 μM) or solvent control (no addition, NA) for 2 h. Cells were immunostained with a VAP rabbit polyclonal antibody and an AlexaFuor488-conjugated secondary antibody, followed by a CERT IgY antibody and an AlexaFluor594-conjugated secondary antibody. Images were captured using a Zeiss Axiovert 200m inverted microscope as described in Materials and Methods.