Real-time quantitative PCR analyses were performed using the SybrH Green reagents kit (Applied Biosystems, Courtaboeuf, France) with an ABI PRISM 7900HT Sequence detector instrument (Applied Biosystems) according to the manufacturer’s instructions. Amplification was carried out in a final volume of 10 mL with 20 ng of reverse transcribed total RNA, 150 nM (HMGCoAR, LDLR) or 300 nM (UBC and SREBF-2) of both sense and antisense primers (Eurogentec, Lie`ge, Belgium) in the SybrH Green PCR Master Mix (MESA GREEN qPCR, Eurogentec). The cycling conditions comprised 40 cycles at 95uC for 15 s and 60uC for 1 min. Relative quantification for a given gene, expressed as fold-variation over control at T0, was calculated after normalization to a reference gene (UBC) and determination of the CT (cycle threshold) difference between conditioned cells and control cells at T0 using the comparative CT method [20]. Efficiencies of the target and control amplification were very similar. Dynamin 2 RNAi Treatment Non targeting siRNA (si scramble) and siRNA against dynamin2 (si Dyn2) (GGA CAU GAU CCU GCA GUUdTdT) were described in [21]. Briefly, HeLa cells were transfected using RNAiMAX (Invitrogen) according to the manufacturer’s instruc- tions. Knockdown of Dyn2 was observed 48 h post-treatment. Cells transfected with scramble siRNA were used as a control. Dynasore Impairs Cholesterol Homeostasis Western Blotting Cells were lysed in 1% triton 6100 in PBS containing protease inhibitors. Proteins (30 mg per lane) were separated on a 3–8% NuPAGE in Tris acetate gels (Invitrogen) and transferred to a PDVF membrane (Bio-Rad, Hercules, CA). The membrane was incubated for 1 h in blocking solution (5% nonfat dry milk, 0.1% Tween 20 in PBS). Dynamin 2 was detected using mouse monoclonal anti-dynamin 2 (Invitrogen). A rabbit anti-b-actin polyclonal antibody was used as loading control (Sigma). Development was performed with chemiluminescent detection kit (ECL, Amersham Life Science). Results Dynasore inhibits the uptake of LDL cholesterol in HeLa cells and human macrophages We tested the effect of dynasore on HeLa cells, where dynamin was first reported to control CT trafficking [9]. Dynasore was tested at 80 mM, the concentration shown to efficiently block the internalization of the transferrin receptor [10]. We measured the uptake of fluorescently labeled human LDL by flow cytometry. Dynasore treatment led to a strong inhibition of LDL uptake at 10 mg/ml with less than 10% of the uptake level measured in untreated cells (Figure 1A). For comparison, the uptake of LDL was decreased by about 50% in our previous study with HeLa cells expressing the K44A inhibitory mutant of dynamin [9]. The continuous uptake of LDL for 6 hours in sterol-starved cells led to a 2.5 fold increase of the total amount of cellular cholesterol – free and esterified – as measured by HPLC in control cells. Despite the strong inhibition of LDL uptake, there was still an increase albeit more moderate in dynasore-treated cells (50615 and 4368 nmol/ Figure 2. Effects of dynasore on the intracellular distribution of FC and LDL in HeLa cells and HMDM. (A) Hela cells were loaded with 200 mg/ml LDL for 24 h. Cells were then treated for 6 h with 80 mM dynasore or without (control) and stained with filipin to detect FC. (B) Cells were treated as described above with 200 mg/ml DiI-LDL. (C) HMDM were incubated for 6 h in LPDS medium containing 50 mg/ml DiI-AcLDL with 80 mM dynasore or without (control). (D) HMDM were loaded with 50 mg/ml DiI-AcLDL for 24 h and then treated for 6 h with 80 mM dynasore or without (control). Images were obtained using wide- field epifluorescence microscopy. Scale bars, 10 mm. doi:10.1371/journal.pone.0029042.g002 Figure 3. Dynasore treatment results in the endolysosomal accumulation of FC and LDL in HeLa cells. Cells were treated for 6 h with 80 mM dynasore or without (control) in medium containing 200 mg/ml LDL (A) or DiI-LDL (B) and processed for filipin staining (A) or DiI-LDL detection (B). Left panels present Lamp1 staining. Merge of Lamp1 with FC (A) or with DiI-LDL (B) is shown in the right panel. Scale bars, 10 mm. doi:10.1371/journal.pone.0029042.g003 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29042