Figure 3. Organization of cholesterol in the PM of ORP2/HeLa cells.

(A) The ORP2/HeLa cell line 21f3 was labelled with [¹⁴C]cholesterol for 42 h in the absence (white bars) or presence (black bars) of 1 μg/ml doxycycline, either in a medium containing 5% LPDS or one supplemented with 10% complete FBS. The cell-surface cholesterol was extracted for 5 min with 5 mM methyl-β-cyclodextrin, and radioactivity in the medium and in the cells was determined. The bars represent the portion recovered in the medium (mean±S.E.M.; n=3). (B) PMs of ORP2/HeLa cells incubated in 10% FBS containing medium in the absence of presence of induction, were enriched using the colloidal silica technique. Cholesterol was quantified by an enzymatic method and phospholipids by LC–ESI–MS, as described in the Experimental section. The results are shown as a mol% distribution, and represent the means±S.E.M. for the three cell lines. (C) The ORP2/HeLa cell lines were labelled with [¹⁴C]cholesterol for 42 h in the absence or presence of induction in a medium supplemented with 10% FBS, and distribution of the radioactivity between Triton X-100 insoluble and soluble fractions was determined as specified in the Experimental section. The bars represent the portion of [¹⁴C]cholesterol in two top fractions containing the DRM, and represent a mean±S.E.M. for three experiments. **P<0.01, *P<0.05, t test.