Dynasore Impairs Cholesterol Homeostasis Figure 7. Dynasore impairs cellular cholesterol efflux from HMDM. HMDM were incubated for 6 h with 100 mg/ml AcLDL with 80 mM dynasore or without (control). The cellular cholesterol efflux to 10 mg/ml apoA-I or 15 mg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels was expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MaˆCD was quantified as above. doi:10.1371/journal.pone.0029042.g007 AcLDL uptake and the lower concentration used (50 mg/ml). Indeed, after 24 hours of incubation with AcLDL, the level of CE in HMDM was higher and represented 40% of total cholesterol (data not shown). Again, dynasore treatment led to a strong decrease (90%) of the amount of CE specifically generated by ACAT (Figure 6C) and of the incorporation of myristate into CE (82% inhibition) (Figure 6D). Together these findings demonstrate that the abnormal endosomal accumulation of LDL induced by dynasore treatment results in a strong defect of FC delivery to the ER. Dynasore decreased cholesterol efflux in human macrophages Although reverse cholesterol transport (RCT) is a general peripheral process, macrophages are the primary cells that are overloaded with cholesterol in atherosclerotic lesions [25]. The efficiency of cholesterol efflux, the first step of RCT from macrophage foam cells, is determined not only by the activity of cholesterol transporters and extracellular acceptors, but also by the availability of cholesterol for efflux. It was therefore interesting to test whether the abnormal sequestration of large amounts of cholesterol in the endolysosomal compartment could affect the cholesterol efflux capacity of HMDM. Thus, we measured cholesterol mass efflux to either lipid-free apoA-I or to HDL (high density lipoproteins) in HMDM loaded with AcLDL (Figure 7A). Dynasore treatment resulted in a strong decrease of cholesterol efflux to apoA-I (40%) and HDL (70%). Similar results were observed when cholesterol efflux was stimulated with the natural LXR/RXR agonists, 22OHC/9cRA (Figure 7B). The two ABC (ATP binding cassette) members of cholesterol transporters, ABCA1 and ABCG1 have been involved in cholesterol efflux to lipid poor apoA-I and HDL, respectively [25,26]. We thus examined the impact of dynasore treatment on ABCA1 and ABCG1 gene expression. As expected, the addition of AcLDL to HMDM led to an increase of ABCA1 and ABCG1 mRNA levels Figure 8. Dynasore treatment affects M6PR distribution. (A) Cells expressing GFP-M6PR were treated 6 h with 80 mM dynasore or without (control). Images were obtained using wide-field epifluores- cence microscopy. Scale bars, 10 mm. doi:10.1371/journal.pone.0029042.g008 PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29042