In theory, this assay could measure esterification of both cellular and LDL cholesterol. However, the augmentation observed upon Pdro (+)-Camphor depletion mostly represents esterification of LDL-derived cholesterol, since in the absence of LDL, cholesterol esterification was rather slightly diminished by the depletion of Pdro. These results strongly suggest that cholesterol flux between LE/LY and the ER is increased when Pdro is depleted. It is also consistent with the absence of cholesterol accumulation in the perinuclear network described in Figure 4A. One mechanism that could contribute to increased cholesterol egress from LE/LY is an enhanced uptake of LDL. To test this hypothesis, control cells or cells depleted for Pdro were incubated with cholesteryl oleate-labeled LDL for 12 hours, and total 10-Undecenoic acid zinc salt radioactivity in cell homogenates was counted. As shown in Fig 5A, the uptake of LDL was significantly increased upon Pdro depletion, suggesting that the increase in free cellular cholesterol is due, at least in part, to an increased LDL uptake followed by an increased cholesterol egress from LE/LY. The LDL preparations used for uptake assay are modified LDL that are internalized through Scavenger Receptors or through non-receptor mediated fluid-phase macropinocytosis. Considering that the plasma membrane protrusions described in Figure 4A are often associated with macropinocytic activity, we investigate whether Pdro knockdown could stimulate macropinocytosis. Control- or siPdrotreated cells were incubated with RITC-conjugated dextran and macropinocytic activity was analyzed by flow cytometry. Figure 5B shows that the amount of fluorescent dextran taken up by the cells was significantly increased upon knockdown of Pdro. Therefore, the absence of Pdro stimulates an increased macropinocytosis that may contribute to LDL uptake. The relative contribution of Scavenger Receptors and macropinocytosis in the increased LDL uptake is currently being investigated. Excess cellular cholesterol is converted into nontoxic cholesteryl ester for storage or removed by cholesterol efflux at the plasma membrane.