the cholesterol loading medium. When ACAT activity is inhibited, CE are provided by pre-existing pools such as endocytosed LDL. Therefore, the difference in cholesterol esterification measured by HPLC with and without Sandoz 58-035 represents the specific amount of cholesterol esterified by ACAT. Cellular cholesterol mass efflux to apoA-I and HDL in cholesterol-loaded macrophages After treatment with dynasore or U18666A, HMDM were equilibrated in RPMI1640 0.2% BSA for 16 h in the presence or not of 5 mg/ml 22-hydroxy-cholesterol (22OH-C) and 10 mM 9-cis retinoic acid (9cRA) (Sigma-Aldrich). Cellular cholesterol efflux to either 1 mg/ml methyl-b-cyclodextrin (MbCD), 10 mg/ml lipid- free apoA-I (Sigma-Aldrich) or 15 mg/ml high-density lipoprotein- phospholipids (HDL-PL isolated from normolipidemic human plasma by preparative ultracentrifugation) was measured during a 4 h chase period. Drugs were maintained during the equilibration and efflux periods. At the end of the efflux, the medium was collected and the cells lysed in 0.2 M NaOH. Cell and media were extracted and analyzed for free and esterified cholesterol mass by HPLC as described above. HDL samples were separately analyzed to allow correction for HDL cholesterol present in relevant media samples. Mass cholesterol efflux is expressed as the percentage of Dynasore Impairs Cholesterol Homeostasis efflux (medium cholesterol over total cholesterol-medium and cells) [18]. Immunofluorescence Analysis of LDL-derived cholesterol trafficking was performed by immunofluorescence as described [9]. Briefly, HeLa cells and HMDM were grown on cover slips for 48 h in LPDS medium. To monitor LDL trafficking, cells were incubated with fluorescent LDL for indicated times. Cells were incubated with 200 mg/ml LDL or 50 mg/ml AcLDL and then stained with filipin (50 mg/ml) to detect FC as described [19]. Cells were fixed in 4% paraformaldehyde (PFA) in PBS. For immunolocalization studies, cells were incubated with primary and secondary antibodies diluted in PBS/BSA with saponin. Cover slips were mounted in MOWIOL, and cells were imaged with an epifluorescent Leica microscope. RNA extraction, RT-PCR and real time quantitative PCR Total RNA was extracted from HeLa cells using the RNeasy Mini kit (Qiagen, Courtaboeuf, France). One microgram of total RNA was transcribed to cDNA using random hexamers (Amersham, Orsay, France) and SuperScriptII reverse transcrip- tase (Invitrogen). Specific primers were described previously [9]. Figure 1. Effect of dynasore on LDL uptake and total cholesterol in HeLa cells and HMDM. Cells were incubated for 4 h with 0–200 mg/ml DiI-LDL (A) or 0–100 mg/ml DiI-AcLDL (C) at 37uC with 0.4% v/v DMSO (control) or 80 mM dynasore. The total amount of endocytosed DiI-LDL or 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 0.4% v/v DMSO (control) or 80 mM dynasore. 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.g001 PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29042