(1.9-fold and 3.5-fold, respectively). However, this up-regulation was completely abolished when HMDM were treated by dynasore (Figure 7C). Finally, we examined whether dynasore treatment could affect the amount of cholesterol associated with the plasma membrane, which is involved in the passive cholesterol efflux pathway. To address this question, we used MbCD, a high-affinity acceptor of cholesterol. Short time incubation with MbCD was shown to remove cholesterol from the plasma membrane [27]. Under these conditions, cholesterol efflux was significantly decreased (34%) in dynasore-treated cells (Figure 7D). Together, these results indicate that dynasore, by affecting intracellular cholesterol trafficking, has a major impact on several pathways implied in active and passive cholesterol efflux. Dynasore affects the intracellular distribution of the mannose 6-phosphate receptor We tested whether dynasore could affect the trafficking of other cargos through the late endosomal network. We therefore studied the cation-independent mannose 6-phosphate receptor (M6PR), which constitutively recycles between late endosomes and the Golgi complex [28]. When HeLa cells expressing a GFP tagged M6PR were treated by dynasore, we observed that the M6PR was not present at the Golgi complex and was localized instead in dispersed endosomal structures (Figure 8). This result, in Dynasore Impairs Cholesterol Homeostasis agreement with a previous study [28], indicates that the effect of dynasore was not restricted to the block of FC from the late endosomal network but affected also cargo trafficking at this interface. Comparison of dynasore and U18666A treatments on cholesterol trafficking in HeLa cells and macrophages These results led us to compare the effects of dynasore with U18666A, a hydrophobic amine that has been extensively used to study cholesterol homeostasis, and more particularly cholesterol intracellular trafficking [29]. In contrast to dynasore, we found that U18666A treatment led to a respective 20% and 40% increase of LDL uptake and AcLDL in HeLa cells and HMDM (Figure 9 A and C). Accordingly, we measured an increase in the total amount of cholesterol present in HeLa cells and HMDM after U18666A treatment (Figures 9 B and D). The inhibition of cholesterol egress from late endosomes and lysosomes is a well- known effect of U18666A [29,30]. As expected, treatment with U18666A led to the formation of swollen endosomal structures loaded with FC in HeLa cells and HMDM (Figure 10). These structures were part of the endolysosomal network as confirmed by staining with Lamp1 (not shown). As a consequence, there was no response of sterol-sensitive genes to the addition of LDL or AcLDL in U18666A treated cells (Figures 11 A and B). In contrast to cells Figure 9. Effect of U18666A on LDL uptake and total cholesterol in HeLa cells and HMDM. LDL uptake was measured in HeLa cells (A) and HMDM (C) after incubation at 37uC for 4 h with 0–200 mg/ml DiI-LDL or 0–100 mg/ml DiI-AcLDL, respectively, with 3 mg/ml U18666A or without (control). The amount of endocytosed DiI-LDL and DiI-AcLDL was measured by flow cytometry. Values represent the mean 6 SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 3 mg/ml U18666A or without (control). Each value is the mean 6 SD of triplicate experiments and expressed as nanomoles per mg of cell proteins. doi:10.1371/journal.pone.0029042.g009 PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29042