CoA:cholesterol acyltransferase (ACAT) enzyme localized in the ER [7]. ACAT rapidly esterifies FC in CE to be stored in cytoplasmic lipid droplets, in response to an increased concentra- tion of cholesterol in the ER. ACAT activity is therefore required for decreasing the amount of cytotoxic FC and for maintaining a low level of cholesterol in the ER membranes, such that minimal variations of sterol concentrations can be sensed. In some cases, ER-derived vesicular structures that are positive for ACAT can also be involved in this process. It has been proposed that these structures, which are distinct from the conventional ER, may protect the non-fragmented ER from being overloaded with cholesterol [8]. The GTPase dynamin is a mechano-chemical enzyme required for the pinching and release of a completed clathrin-coated pit from the plasma membrane. In HeLa cells expressing the K44A inactivated form of dynamin, we have revealed a new role for dynamin in the delivery of endolysosomal FC to the ER [9]. The inactivation of dynamin led to a Niemann-Pick type C-like phenotype (NPC) with the accumulation of swollen late endo- somes/lysosomes (LE/LS) engorged with FC. Dynamin inactiva- tion was also associated with a strong reduction of sterol-sensitive genes regulation and a decrease of the esterification of the intracellular pool of cholesterol by ACAT. If K44A HeLa cells are an easily amenable cell model, we sought to study the role of dynamin in more physiologically relevant cell types such as macrophages, which play a key role in atherosclerosis. However there are technical limitations to obtain macrophages expressing the inactivated form of dynamin. We therefore took advantage of the membrane permeant chemical compound, dynasore, a recently described noncompetitive inhibitor of the GTPase activity of dynamin [10,11]. Dynasore presents also the advantage to act within minutes and to have a completely reversible activity. To validate the use of this new drug, we studied the impact of dynasore on critical steps of cholesterol trafficking in both HeLa cells and HMDM. We show here that dynasore rapidly inhibits the egress of free cholesterol from the endolysosomal network in these two cell types. As a result, the sterol-sensitive gene regulation of cholesterol homeostasis is inhibited. Dynasore is therefore a new drug that will be useful for further understanding cholesterol endosomal trafficking, a key step in cholesterol homeostasis. Materials and Methods Cells and reagents HeLa cells were cultured in Dulbecco modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen, Cergy-Pontoise, France), 2 mM glutamine, 100 UI/ ml penicillin, 100 mg/ml streptomycin (Sigma-Aldrich, St Louis, MO). Cells were treated with dynasore (Sigma-Aldrich) at 80 mM in the presence of 1% lipoprotein-deficient serum (LPDS) medium to avoid inactivation of dynasore by serum proteins. U18666A (Sigma-Aldrich) was used at 3 mg/ml in 1% LPDS medium. Monocytes were isolated from human blood of normolipidemic donors by ficoll density gradient (Eurobio, les Ulis, France) and subsequently differentiated into human monocytes-derived mac- rophages (HMDM) by adhesion on plastic Primaria plates (Falcon BD, Franklin Lakes, NJ,) for 7 days in RPM1640 medium, supplemented with 10% heat-inactived FCS, 2 mM glutamine, 100 UI/ml penicillin, 100 mg/ml streptomycin, and 20 ng/ml hM-CSF (human macrophages colony-stimulating factor) (AbCys, Paris, France). HeLa cells expressing a GFP tagged M6PR were described in [12]. Cells were cultured in Dulbecco modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) Dynasore Impairs Cholesterol Homeostasis (Invitrogen,), 2 mM glutamine, 100 UI/ml penicillin, 100 mg/ml streptomycin (Sigma-Aldrich,) completed with geneticin. They were treated with dynasore the same way that HeLa cells. Antibodies The mouse anti-human Lamp-1 antibody was from BD Transduction Laboratories. Primary antibodies were revealed by incubation with Alexa FluorH488 donkey anti-mouse IgG (Invitrogen). Cholesterol delivery to the cell Human LDL (d = 1.019–1.063 g/ml) were separated from fresh plasma by sequential ultracentrifugation as described previously [9]. HeLa cells deprived of sterols for 48 h in 10% LPDS medium were incubated for the indicated times with LDL (200 mg/ml), supplemented with 50 mM pravastatin and 50 mM sodium mevalonate. HMDM were incubated with 50 or 100 mg/ml of acetylated LDL (AcLDL) prepared using the standard acetic anhydride-sodium acetate solution on ice as described [13]. LDL and AcLDL labeling LDL were labeled with DiI (1,19-dioctadecyl-3,3,3939-tetra- methyl-indocarbocyanine) as described previously [14]. Briefly, DiI were added to LDL to a final ratio of 300 mg for 1 mg LDL protein. After incubation for 18 h at 37uC under nitrogen and light protection, DiI-labeled LDL were isolated by ultracentrifu- gation and extensively dialyzed at 4uC against PBS (Eurobio). For DiI-AcLDL production, AcLDL were first labeled with DiI and acetylated. LDL uptake assay LDL uptake was described in [9]. Briefly, cells grown in LPDS medium during 48 h were washed in PBS and incubated at 37uC with increasing concentrations of DiI-LDL (HeLa cells) or DiI- AcLDL (HMDM) as indicated. After 4 h, cells were treated by trypsin to remove cell surface bound fluorescent LDL. Cells were detached and washed twice in PBS at 4uC. The fluorescence of internalized DiI-LDL or DiI-AcLDL was measured by flow cytometry (emission at 585 nm) and expressed as mean fluores- cence intensity. Measurement of cellular cholesterol After 48 h of culture in LPDS medium, cells were incubated for 6 h with 200 mg/ml LDL (HeLa cells) or 50 mg/ml AcLDL (HMDM) and then lysed in 0.2 M NaOH. Total cholesterol was extracted with methanol (2.5 ml) followed by hexane (5 ml). About 4.5 ml of the hexane phase was evaporated under vacuum and dissolved in mobile phase. Separation of FC and CE including cholesteryl docohexanoate (CDH), cholesteryl arachidonate (CA), cholesteryl linoleate (CL), cholesteryl myristate (CM), cholesteryl oleate (CO) and cholesteryl stearate (CS) was done by reverse phase HPLC on a C-18 column (2560.46 cm length, 5-mm pore size, Sigma-Aldrich) by measuring the 205 nm absorbance after elution with acetonitrile/isopropanol (30/70, v/v) [15,16]. The amount of proteins present in cell lysates and LDL preparations was measured using the bicinchoninic acid (BCA) method [17]. ACAT activity 120 mM sodium myristate (Sigma-Aldrich) was added during cholesterol delivery to the cells. The ACAT enzyme activity was evaluated by quantifying the incorporation of myristate into cholesteryl myristate by HPLC as described above. The ACAT inhibitor Sandoz 58-035 (Sigma-Aldrich) (10 mg/ml) was added to PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29042