Fig. 5.
CD-induced cholesterol accumulation in the media depends on serum lipoproteins. U2OS-SRAshNPC1 cells were loaded overnight with d7-acLDL in the presence of lalistat and then treated with CD or vehicle for 6 h in media of differing compositions. A: After treatment in media containing FBS, lipoproteins were isolated from conditioned media and assayed for d7 cholesterol content. Sypro Ruby-stained native PAGE gel analysis (left) and d7 cholesterol content (right) of conditioned media and its lipoprotein fractions (F1 and F2) and remaining material (F3) after lipoprotein isolation. Pure hLDL and hHDL are shown as markers. B: Media-associated d7 cholesterol quantified after 6 h of treatment in FBS, LPDM, or LPDM supplemented with 25 μg/ml APOA1, HDL, LDL, or BSA. Efflux reported relative to efflux with FBS. C: PM-localized lysosomal cholesterol after 6 h of CD treatment in FBS or LPDM reported as a percentage of the total cholestenone pool that was deuterated. D: Reesterification of lysosomal cholesterol after 6 h of CD treatment in FBS or LPDM. E: Esterification of nondeuterated cholesterol (d9 CE 18:1) after 6 h of CD treatment in FBS or LPDM. Means ± SEs (n = 3–5). *P < 0.05 by unpaired t-test or two-way ANOVA for comparisons indicated. hHDL, human HDL; hLDL, human LDL; V, vehicle.