Dynasore Impairs Cholesterol Homeostasis of cholesterol homeostasis [2]. After 48 hours of sterol starvation, cells were incubated with LDL in the presence or in the absence of dynasore. The kinetics of expression of the LDLR gene were monitored by real time quantitative RT-PCR analysis in HeLa cells (Figure 5A). As expected in cells that are in excess of exogenous cholesterol, a potent repression of the expression of the LDLR gene was measured as early as 5 hours after the addition of LDL and for as long as 24 hours in control cells. In contrast, no down-expression of the LDLR gene could be measured in cells treated with dynasore at similar times, and LDLR expression levels were similar to those observed in cells not supplemented with LDL. Seven hours after LDL addition, however, the effect of dynasore decreased progressively and the level of LDLR gene expression resumed to control values at 24 hours. Thus, we chose to analyze the effects of dynasore after 6 hours of LDL addition, which corresponds to a strong repression of the sterol-sensitive transcriptional response in control cells and to the maximal inhibition by dynasore. At 6 hours, dynasore also strongly inhibited the LDL-induced repression of another major sterol- sensitive gene, HMGCoAR, whereas the repression on SREBF-2 gene expression was not observed at this time in HeLa cells (Figure 5B). Similar results were obtained in dynasore-treated HMDM, with a strong inhibition of the down-regulation of sterol- sensitive genes (LDLR, HMGCoAR and to a lesser extent, SREBF-2) induced by AcLDL (Figure 5C). These data are in agreement with the accumulation of LDL and AcLDL in enlarged Figure 5. Dynasore blocks sterol-sensitive genes regulation in HeLa cells and HMDM. (A) Kinetics of LDLR expression analyzed by RT-PCR. HeLa cells were grown in LPDS medium for 48 h and further incubated for the indicated times with medium containing either LPDS, 200 mg/ml LDL, or 200 mg/ml LDL with 80 mM dynasore. (B) The expression level of sterol-sensitive genes (LDLR, HMGCoAR and SREBF-2) was quantified after 6 h in HeLa cells grown in LPDS, with 200 mg/ml LDL or 200 mg/ml LDL with 80 mM dynasore, as indicated. (C) The same experiment was performed in HMDM with 50 mg/ml AcLDL. Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells or HMDM was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate. doi:10.1371/journal.pone.0029042.g005 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29042