endosomes observed in both cell types (Figure 2) and are likely to reflect a defect in the delivery of LDL-derived cholesterol to the ER. Dynasore prevents the esterification of exogenous LDL- derived cholesterol We next measured the fraction of esterified cholesterol by the ACAT enzyme as a marker of the amount of FC being delivered to the ER. Indeed, CE are generated from FC by the activity of the ACAT enzyme [7]. Since this enzyme is strictly localized in the ER membranes, the amount of cholesterol esterified by ACAT reflects the amount of FC delivery to the ER [23,24]. Thus, we measured by HPLC the balance between the pools of free and esterified intracellular cholesterol. When HeLa cells were grown under sterol starvation, cholesterol was mainly detected as FC and esters represented less than 5% of total cholesterol (Figure 6A). After the addition of LDL, the total amount of CE represented more than 30% of total cholesterol. Dynasore treatment reduced this amount by about 15%. This moderate inhibition disagrees with the complete absence of sterol-sensitive genes repression in dynasore treated cells as observed above. Thus, we studied Dynasore Impairs Cholesterol Homeostasis whether a fraction of the measured pool of CE may be independent from the ER-ACAT activity. Indeed, dynasore treatment leads to the abnormal endosomal accumulation of LDL, which are unlikely to be de-esterified by the lysosomal hydrolases and thus could contribute to the total intracellular pool of CE. Therefore, we measured the total amount of CE in HeLa cells in which ACAT activity was pharmacologically inhibited. Under this condition, we found that the amount of CE generated by ACAT accounted for only 38% of the total intracellular pool of CE (Figure 6A). When cells were treated with dynasore, this amount decreased to about 10%, which represents a 74% inhibition of LDL-derived cholesterol esterification. We could confirm this result by measuring the synthesis of cholesteryl myristate by ACAT, an ester that was not initially present in our cells. After addition of myristate, we found by HPLC that the production of cholesteryl myristate was decreased by 80% in cells treated with dynasore (Figure 6B). In HMDM, dynasore treatment decreased the total amount of CE more efficiently than in HeLa cells (Figure 6C). However, the level of CE was initially lower in HMDM than in HeLa cells (11% versus 35%, respectively). This could be due to different kinetics of Figure 6. Dynasore decreases the production of cholesterol esters from LDL- or AcLDL-derived cholesterol. HeLa cells or HMDM were respectively incubated with 200 mg/ml LDL (A) or 50 mg/ml AcLDL (C) and treated for 6 h with 80 mM dynasore or without (control). The total amount of CE was quantified and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 mg/ml ACAT inhibitor (grey bars). The production of cholesteryl myristate was measured in HeLa cells (B) or HMDM (D) treated or not (control) with 80 mM dynasore. Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments. doi:10.1371/journal.pone.0029042.g006 PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29042