Figure 5.

Knockdown of ORP9L expression does not affect oxysterol-regulated SM synthesis. (A) CHO cells cultured in medium A were transiently transfected with siOSBP (75 nM), siORP9L (75 nM), siOSBP and siORP9L (75 nM each), or a nontargeting control siNT (75 nM) for 48 h. Cells then received serine-free medium A containing no addition () or 25-hydroxycholesterol (2.5 μg/ml; ■) for 2 h followed by pulse-labeling with [³H]serine (10 μCi/ml) for 2 h. [³H]Serine-labeled SM, GlcCer and ceramide were extracted from cells, separated by thin-layer chromatography, and quantified as previously described (Perry and Ridgway, 2006). Results are the mean and SE for three separate experiments. (B) Expression of OSBP and ORP9L in CHO cells transiently transfected with control and targeting siRNAs. (C) CHO cells expressing ORP9L under the control of the Tet repressor were cultured in medium A without (−Dox) or with doxycycline (+Dox, 1 μg/ml) for 24 h. Cells then received solvent control () or 25OH (■) and were pulse-labeled with [³H]serine as described in A. Results are the mean and SEM for three separate experiments.