Fig. 3.
Released d7 cholesterol is not detected in isolated EVs. U2OS-SRAshNPC1 cells were loaded overnight with d7-acLDL in the presence of lalistat and then treated for 6 h in media with 500 μM CD, 5 μM ionomycin, or vehicle (H2O for CD and DMSO for ionomycin). A: LC/MS/MS quantification of d7 cholesterol associated with the media over time normalized to cellular protein. B: PM d7 cholestenone over time normalized to cellular protein. C–E: EVs were isolated from conditioned media at 6 h by differential centrifugation. Immunoblot analysis of total cell lysates (cells) and EVs for exosomal marker CD63 and nuclear marker LMB (C). Total d7 cholesterol in 27 ml conditioned media (input), 27 ml EV-depleted media, and EVs (from 27 ml conditioned media) at 6 h (D). Total EV cholesterol normalized to EV protein (E). Means ± SEs (n = 3). *P < 0.05 by unpaired t-test. LMB, lamin B; V, vehicle.