Figure 4. Effect of ORP2 overexpression on the efflux of newly synthesized cholesterol to methyl-β-cyclodextrin.

Control (Ctrl) CHO and ORP2 expressing A5a9 (ORP2/CHO) cells were cultured in lipoprotein-deficient medium containing [¹⁴C]cholesterol as described in the Experimental section. To measure the appearance of newly synthesized [³H]cholesterol at the PM, cells were labelled with [³H]acetate for 15 min and chased for 2 h. During the last 5 min of the chase the cells were incubated with 5 mM methyl-β-cyclodextrin. The synthesized [³H]cholesterol and the pre-existing [¹⁴C]cholesterol in the cells and the media were isolated by HPTLC and HPLC and quantified by liquid-scintillation counting. The results are shown as the percentage of the total [³H] (black bars) or [¹⁴C] (white bars) cholesterol found in the efflux medium (mean±S.E.M.; n=7; ***P<0.001, t test).