mg cell protein, respectively; Figure 1B). This is in agreement with our findings in HeLa cells that cholesterol can enter cells through other dynamin-independent endocytic pathways [9]. We next characterized dynasore in HMDM since they play a central role in the formation and progress of atherosclerotic plaques [22]. The uptake of AcLDL in HMDM was twice as much less efficient than the uptake of LDL in HeLa cells (Figure 1C). The effect of dynasore was less important in HMDM since the uptake of AcLDL uptake was decreased by 50% at 10 mg/ml and by 17% at 100 mg/ml. However, the absolute amount of endocytosed cholesterol was similar in HeLa cells and HMDM treated by dynasore. In agreement with the lower inhibition of AcLDL uptake, the measure of the total amount of cholesterol revealed no difference between control and dynasore-treated HMDM (Figure 1D). Dynasore blocks the egress of free cholesterol from the late endosomal network in HeLa cells and human macrophages We examined the intracellular distribution of FC derived from endocytosed LDL using filipin, a naturally fluorescent antibiotic that binds selectively to the FC fraction present in cellular membranes. We also visualized the intracellular distribution of DiI-LDL since we showed previously that DiI-LDL undergo the same endocytic and degradative processes than unlabeled LDL [9]. Cells were incubated with LDL for 24 hours to allow their distribution within the different endosomal compartments along the endocytic pathway. In control cells, FC was found into small endosomal structures within the cytoplasm of the cell (Figure 2A). A similar pattern was found for DiI-LDL (Figure 2B). Upon dynasore treatment, we could observe the accumulation of FC and Internalized DiI-LDL into numerous enlarged endosomes (Figures 2 A and B). We also examined the intracellular distribution of DiI-AcLDL in HMDM after 6 hours of internal- ization. Dynasore treatment led also to an increase of both the number and the size of the endosomal structures loaded with DiI- AcLDL (Figure 2C). This effect was enhanced when cells were first incubated with DiI-AcLDL for 24 hours before the addition of dynasore (Figure 2D). These endosomes were part of the late endosomal network since they were positive for the lysosomal associated membrane protein 1 (Lamp1), a marker of late endosomes and lysosomes (Figures 3 A and B). Thus, dynasore which blocks the GTPase activity of dynamin, causes the accumulation of endocytosed LDL-derived cholesterol in the late endocytic compartment and prevents its egress from this compartment in both HeLa and HMDM cells. This is in agreement with our previous study showing that dynamin controls the delivery of cholesterol from late endosomes to the ER in HeLa cells [9]. The abnormal endosomal accumulation of LDL was already observed after 15 min of dynasore treatment indicating that dynasore acts at the endoslysosomal level in the same order of time that it requires to inhibit the uptake of transferrin and LDL at the plasma membrane (data not shown) [10]. Down-expression of dynamin results in the inhibition of free cholesterol egress from the late endosomal network To confirm the specificity of dynasore treatment, we depleted endogenous dynamin 2, the major dynamin isoform expressed in HeLa cells using specific siRNA. Dyn2 siRNa treatment led to a strong down-expression of dynamin 2 as assessed by real time quantitative RT-PCR (data not shown) and western blot analysis (Figure 4A). Under this treatment, there was an accumulation of swollen endosomal structures loaded with LDL or FC, and positive Dynasore Impairs Cholesterol Homeostasis Figure 4. Dynamin silencing leads to endolysosomal accumu- lation of FC and LDL in HeLa cells. HeLa cells were transfected with Dyn2 siRNA or scramble siRNA (control) for 48 h in LPDS medium (LPDS) or LPDS medium and 200 mg/ml LDL (LDL). Dyn2 and actin levels were determined by western blot (A). Cells were loaded for 6 h with 200 mg/ml LDL and stained with filipin to detect FC (C). Cells were treated as described above with 200 mg/ml DiI-LDL (B). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 mm. doi:10.1371/journal.pone.0029042.g004 for Lamp1 (Figure 4 B and C). These results faithfully reproduce the phenotype observed with dynasore treatment and therefore exclude dynasore side effects at the endosomal interface. Dynasore prevents the down-regulation of sterol- sensitive genes induced by exogenous LDL-derived cholesterol The delivery of FC from the late endolysosomal compartment to the ER is a key process in the transcriptional regulation of sterol-sensitive genes [1,7,9]. We therefore studied whether dynasore had an impact on this regulation. In contrast to the conditional K44A HeLa cell line that requires 48 hours to express the K44A dynamin mutant [9], dynasore is active within a few minutes. We thus determined the minimal amount of time required to measure an effect of dynasore on the expression of the LDLR gene, one of the major actors of the transcriptional control PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29042