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 µg/ml DiI-LDL (A) or 0–100 µg/ml DiI-AcLDL (C) at 37°C with 0.4% v/v DMSO (control) or 80 µM dynasore. The total amount of endocytosed DiI-LDL or DiI-AcLDL was measured by flow cytometry. Values represent the mean ± 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 µM dynasore. Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.
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 µg/ml LDL for 24 h. Cells were then treated for 6 h with 80 µM dynasore or without (control) and stained with filipin to detect FC. (B) Cells were treated as described above with 200 µg/ml DiI-LDL. (C) HMDM were incubated for 6 h in LPDS medium containing 50 µg/ml DiI-AcLDL with 80 µM dynasore or without (control). (D) HMDM were loaded with 50 µg/ml DiI-AcLDL for 24 h and then treated for 6 h with 80 µM dynasore or without (control). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 µm.
Figure 3.
Dynasore treatment results in the endolysosomal accumulation of FC and LDL in HeLa cells.
Cells were treated for 6 h with 80 µM dynasore or without (control) in medium containing 200 µg/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 µm.
Figure 4.
Dynamin silencing leads to endolysosomal accumulation of FC and LDL in HeLa cells.
HeLa cells were transfected with Dyn2 siRNA or scramble siRNA (control) for 48 h in LPDS medium (LPDS) or LPDS medium and 200 µg/ml LDL (LDL). Dyn2 and actin levels were determined by western blot (A). Cells were loaded for 6 h with 200 µg/ml LDL and stained with filipin to detect FC (C). Cells were treated as described above with 200 µg/ml DiI-LDL (B). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 µm.
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 µg/ml LDL, or 200 µg/ml LDL with 80 µM 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 µg/ml LDL or 200 µg/ml LDL with 80 µM dynasore, as indicated. (C) The same experiment was performed in HMDM with 50 µg/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.
Figure 6.
Dynasore decreases the production of cholesterol esters from LDL- or AcLDL-derived cholesterol.
HeLa cells or HMDM were respectively incubated with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (C) and treated for 6 h with 80 µM 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 µg/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 µM dynasore. Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.
Figure 7.
Dynasore impairs cellular cholesterol efflux from HMDM.
HMDM were incubated for 6 h with 100 µg/ml AcLDL with 80 µM dynasore or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels was expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.
Figure 8.
Dynasore treatment affects M6PR distribution.
(A) Cells expressing GFP-M6PR were treated 6 h with 80 µM dynasore or without (control). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 µm.
Figure 9.
Effect of U18666A on LDL uptake and total cholesterol in HeLa cells and HMDM.
LDL uptake was measured in HeLa cells (A) and HMDM (C) after incubation at 37°C for 4 h with 0–200 µg/ml DiI-LDL or 0–100 µg/ml DiI-AcLDL, respectively, with 3 µg/ml U18666A or without (control). The amount of endocytosed DiI-LDL and DiI-AcLDL was measured by flow cytometry. Values represent the mean ± SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 3 µg/ml U18666A or without (control). Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.
Figure 10.
Effects of U18666A on the intracellular distribution of FC and LDL in HeLa cells and HMDM.
HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/mL LDL (A) or 50 µg/ml AcLDL (C) with 3 µg/ml U18666A or without (control) and stained with filipin to detect FC. (B–D) HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/ml DiI-LDL (B) or 50 µg/ml DiI-AcLDL (D) with 3 µg/ml U18666A or without (control) and processed to visualize LDL distribution. Images were obtained using wide-field epifluorescence microscopy.
Figure 11.
U18666A inhibits ACAT activity and sterol-sensitive genes regulation in HeLa cells and HMDM.
Cells were grown in LPDS medium for 48 h and further incubated for 6 h with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (B) with 3 µg/ml U18666A or without (control). Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells (A) or HMDM (B) was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate. The total amount of CE was quantified HeLa cells (C) and in HMDM (D) and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). Cholesteryl myristate formation was measured in HeLa cells (E) or HMDM (F) with 3 µg/ml U18666A or without (control). Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.
Figure 12.
U18666A impairs cellular cholesterol efflux from HMDM.
Cells were incubated with 100 µg/ml AcLDL for 6 h and treated with 3 µg/ml U18666A or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.