lpds  (Millipore)

 
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  • 94
    Name:
    Lipoprotein Deficient Serum from fetal calf
    Description:

    Catalog Number:
    S5394
    Price:
    None
    Applications:
    Studies show that the production of sterols is reduced in human fibroblasts cultured in the presence of lipoprotein deficient serum.
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    Structured Review

    Millipore lpds
    Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in <t>LPDS</t> followed by <t>transfection</t> with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .

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    Images

    1) Product Images from "Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction"

    Article Title: Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0686-09.2009

    Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in LPDS followed by transfection with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .
    Figure Legend Snippet: Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in LPDS followed by transfection with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .

    Techniques Used: Transfection, Staining, Mutagenesis

    2) Product Images from "Membrane cholesterol as regulator of human rhomboid protease RHBDL4"

    Article Title: Membrane cholesterol as regulator of human rhomboid protease RHBDL4

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002640

    Identification of potential cholesterol-binding motifs in RHBDL4. A, human RHBDL4 protein sequence is displayed with the six transmembrane regions highlighted in gray (determined by homology with GlpG). Furthermore, potential CRAC motifs and the mirror version ( CARC ) are highlighted. B, ). Serine 144 and histidine 195 are displayed as sticks and form the active center. Tyr-106 in TMS3 and Tyr-205 in TMS6 are highlighted. C, comparison of RHBDL4 sequences from a total of 72 species (11 examples displayed here) show a high degree of conservation across species for the CRAC motif in TMS6. D, point mutations in RHBDL4 increase protease activity. Tyr-106 and Tyr-205 were mutated to alanine. RHBDL4 mutants were transfected into HEK 293T cells, and the generation of endogenous APP CTFs was determined by Western blot analysis. Representative Western blotting of 7–9 independent experiments is displayed. F, analysis of APP mRNA levels upon transfection of RHBDL4 WT, Y106A, Y205A, and Y106A/Y205A. No significant differences for APP mRNA were detected. GAPDH and β-actin served as reference genes. Data are displayed as mean expression ± S.E., n = 3. One-way analysis of variance followed by Dunnett's post hoc comparison with WT as control was performed. G, RHBDL4 mutants are not responsive to LPDS treatment. 12 h post-transfection, HEK 293T cells were treated with either 10% FCS or LPDS in DMEM for 24 h. A representative Western blotting of six independent experiments is shown. E and H, APP CTFs or APP full length were quantified, and results were normalized to β-actin and then either compared with WT ( D ) or FCS treatment ( F ). D, WT was set to 1 and is indicated by the blue dashed line , and F, FCS-treated samples were set to 1. Mean ± S.E. are displayed, n = 6–9 as stated above, p values for Holm-Bonferroni corrected one sample t test are reported. I, pulldown of RHBDL4 with biotinylated cholesterol. RHBDL4 WT and mutants were transfected, and membrane preparations were incubated with alkyne-cholesterol. After clicking biotin-azide to cholesterol, a neutravidin pulldown was performed. Total cell lysates were collected before the click chemistry was performed, and the Western blotting shows expression of RHBDL4 WT and mutants. Representative Western blots of 3–4 independent experiments are shown. D, F, and I, detection of APP full length and endogenous CTFs with Y188, RHBDL4 with anti-Myc antibody, and β-actin as a loading control.
    Figure Legend Snippet: Identification of potential cholesterol-binding motifs in RHBDL4. A, human RHBDL4 protein sequence is displayed with the six transmembrane regions highlighted in gray (determined by homology with GlpG). Furthermore, potential CRAC motifs and the mirror version ( CARC ) are highlighted. B, ). Serine 144 and histidine 195 are displayed as sticks and form the active center. Tyr-106 in TMS3 and Tyr-205 in TMS6 are highlighted. C, comparison of RHBDL4 sequences from a total of 72 species (11 examples displayed here) show a high degree of conservation across species for the CRAC motif in TMS6. D, point mutations in RHBDL4 increase protease activity. Tyr-106 and Tyr-205 were mutated to alanine. RHBDL4 mutants were transfected into HEK 293T cells, and the generation of endogenous APP CTFs was determined by Western blot analysis. Representative Western blotting of 7–9 independent experiments is displayed. F, analysis of APP mRNA levels upon transfection of RHBDL4 WT, Y106A, Y205A, and Y106A/Y205A. No significant differences for APP mRNA were detected. GAPDH and β-actin served as reference genes. Data are displayed as mean expression ± S.E., n = 3. One-way analysis of variance followed by Dunnett's post hoc comparison with WT as control was performed. G, RHBDL4 mutants are not responsive to LPDS treatment. 12 h post-transfection, HEK 293T cells were treated with either 10% FCS or LPDS in DMEM for 24 h. A representative Western blotting of six independent experiments is shown. E and H, APP CTFs or APP full length were quantified, and results were normalized to β-actin and then either compared with WT ( D ) or FCS treatment ( F ). D, WT was set to 1 and is indicated by the blue dashed line , and F, FCS-treated samples were set to 1. Mean ± S.E. are displayed, n = 6–9 as stated above, p values for Holm-Bonferroni corrected one sample t test are reported. I, pulldown of RHBDL4 with biotinylated cholesterol. RHBDL4 WT and mutants were transfected, and membrane preparations were incubated with alkyne-cholesterol. After clicking biotin-azide to cholesterol, a neutravidin pulldown was performed. Total cell lysates were collected before the click chemistry was performed, and the Western blotting shows expression of RHBDL4 WT and mutants. Representative Western blots of 3–4 independent experiments are shown. D, F, and I, detection of APP full length and endogenous CTFs with Y188, RHBDL4 with anti-Myc antibody, and β-actin as a loading control.

    Techniques Used: Binding Assay, Sequencing, Activity Assay, Transfection, Western Blot, Expressing, Incubation

    3) Product Images from "Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿"

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05423-11

    Inhibition of LDs biosynthesis in HBV-producing cells. (A) HepG2 2.2.15 cells were grown for 24 h in complete medium containing either FBS or LPDS. LDs were stained with Bodipy 493/503 and visualized using a Nikon E600 fluorescence microscope. (B) HepG2
    Figure Legend Snippet: Inhibition of LDs biosynthesis in HBV-producing cells. (A) HepG2 2.2.15 cells were grown for 24 h in complete medium containing either FBS or LPDS. LDs were stained with Bodipy 493/503 and visualized using a Nikon E600 fluorescence microscope. (B) HepG2

    Techniques Used: Inhibition, Staining, Fluorescence, Microscopy

    HBV secretion from LPDS-treated cells. HepG2 2.2.15 cells were grown for 24 h in complete medium supplemented with 10% FBS, LPDS, or a mixture of the two. Where indicated, Lova or Cpz was added as a control. (A) The amount of HBV DNA secreted by an equal
    Figure Legend Snippet: HBV secretion from LPDS-treated cells. HepG2 2.2.15 cells were grown for 24 h in complete medium supplemented with 10% FBS, LPDS, or a mixture of the two. Where indicated, Lova or Cpz was added as a control. (A) The amount of HBV DNA secreted by an equal

    Techniques Used:

    Evaluation of the L protein topology in purified microsomes. Cells were grown in complete medium supplemented with 10% FBS or LPDS for 48 h. Following treatment, microsomes were purified from an equal number of cells, split into three aliquots, and treated
    Figure Legend Snippet: Evaluation of the L protein topology in purified microsomes. Cells were grown in complete medium supplemented with 10% FBS or LPDS for 48 h. Following treatment, microsomes were purified from an equal number of cells, split into three aliquots, and treated

    Techniques Used: Purification

    LPDS treatment results in rapid decrease of cholesterol levels in HepG2 2.2.15 cells. Cells were grown for 24 h (A and C) or the times indicated (B) in complete medium supplemented with 10% FBS (considered untreated), 10% LPDS, or a mixture of the two.
    Figure Legend Snippet: LPDS treatment results in rapid decrease of cholesterol levels in HepG2 2.2.15 cells. Cells were grown for 24 h (A and C) or the times indicated (B) in complete medium supplemented with 10% FBS (considered untreated), 10% LPDS, or a mixture of the two.

    Techniques Used:

    HBV replication and viral envelope protein biosynthesis in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for 24, 48, and 72 h. When indicated, 3TC treatment was included as a control of replication
    Figure Legend Snippet: HBV replication and viral envelope protein biosynthesis in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for 24, 48, and 72 h. When indicated, 3TC treatment was included as a control of replication

    Techniques Used:

    Quantification of intracellular nucleocapsids and enveloped virions in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for the times indicated. Cell lysate levels were normalized to the total protein
    Figure Legend Snippet: Quantification of intracellular nucleocapsids and enveloped virions in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for the times indicated. Cell lysate levels were normalized to the total protein

    Techniques Used:

    4) Product Images from "Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells [S]"

    Article Title: Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M043299

    ER stress reduces cholesterol efflux. HepG2 cells were trace-labeled with 3 H-cholesterol. Cholesterol efflux was measured for 24 h in media containing 2 mg/ml fatty acid-free BSA and 10 µg/ml apoA-I with or without 0.1 µM thapsigargin or 5 µM of the LXR agonist T0901317 (A). Alternatively, cholesterol efflux measurements were performed in media containing 10% LPDS with or without thapsigargin or T0901317 (B). Under both conditions, thapsigargin treatment reduces cholesterol efflux, even in the presence of the synthetic LXR agonist. Data show means ± SD from three experiments.
    Figure Legend Snippet: ER stress reduces cholesterol efflux. HepG2 cells were trace-labeled with 3 H-cholesterol. Cholesterol efflux was measured for 24 h in media containing 2 mg/ml fatty acid-free BSA and 10 µg/ml apoA-I with or without 0.1 µM thapsigargin or 5 µM of the LXR agonist T0901317 (A). Alternatively, cholesterol efflux measurements were performed in media containing 10% LPDS with or without thapsigargin or T0901317 (B). Under both conditions, thapsigargin treatment reduces cholesterol efflux, even in the presence of the synthetic LXR agonist. Data show means ± SD from three experiments.

    Techniques Used: Labeling

    ER stress represses cholesterol synthesis. A: HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 0, 4, 8, or 24 h. Afterward, incorporation of 14 C-acetate into free and esterified cholesterol was measured for 1 h. One representative experiment out of three independent experiments is shown. B: HepG2 cells were incubated with media containing 10% LPDS for 24 h. Afterward, cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for another 16 h. HMG-CoA reductase activity was measured as described in the experimental procedures section. De novo synthesis of free and esterified cholesterol as well as HMG-CoA reductase activity decreases after thapsigargin treatment, even when cells were treated with 10 µM lovastatin to induce HMG-CoA reductase activity. Data show means ± SD from three experiments.
    Figure Legend Snippet: ER stress represses cholesterol synthesis. A: HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 0, 4, 8, or 24 h. Afterward, incorporation of 14 C-acetate into free and esterified cholesterol was measured for 1 h. One representative experiment out of three independent experiments is shown. B: HepG2 cells were incubated with media containing 10% LPDS for 24 h. Afterward, cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for another 16 h. HMG-CoA reductase activity was measured as described in the experimental procedures section. De novo synthesis of free and esterified cholesterol as well as HMG-CoA reductase activity decreases after thapsigargin treatment, even when cells were treated with 10 µM lovastatin to induce HMG-CoA reductase activity. Data show means ± SD from three experiments.

    Techniques Used: Incubation, Activity Assay

    ER stress increases SREBP-2 activity. A: HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression of the established SREBP-2 target genes LDL-receptor and HMGCR was determined by qRT-PCR and normalized to 18s expression. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing the SREBP response element. After 4 h, cells were incubated in media containing 10% FBS or 10% LPDS or 10% LPDS plus 0.1 µM thapsigargin for another 24 h. Promoter activity was determined using dual luciferase assay. Withdrawal of exogenous cholesterol by LPDS treatment increases SREBP-2 activity, which is further increased in response to thapsigargin treatment. Data show one representative experiment out of three independently performed experiments.
    Figure Legend Snippet: ER stress increases SREBP-2 activity. A: HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression of the established SREBP-2 target genes LDL-receptor and HMGCR was determined by qRT-PCR and normalized to 18s expression. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing the SREBP response element. After 4 h, cells were incubated in media containing 10% FBS or 10% LPDS or 10% LPDS plus 0.1 µM thapsigargin for another 24 h. Promoter activity was determined using dual luciferase assay. Withdrawal of exogenous cholesterol by LPDS treatment increases SREBP-2 activity, which is further increased in response to thapsigargin treatment. Data show one representative experiment out of three independently performed experiments.

    Techniques Used: Activity Assay, Incubation, Expressing, Quantitative RT-PCR, Transfection, Luciferase

    ER stress impairs apoA-I expression. HepG2 cells were incubated with increasing concentrations of thapsigargin (A, B) or 0.1 µM thapsigargin (C) in media containing 10% LPDS for 24 h. ApoA-I mRNA was determined by qRT-PCR and normalized to 18s expression (A). ApoA-I protein expression was analyzed by Western blotting (B) and immunofluorescence analyses (C). Green: ABCA1; blue: DAPI. Bar = 10 µm. apoA-I mRNA and protein are decreased by thapsigargin treatment. qRT-PCR: mean ± SD (n = 3). Western blot and immunofluorescence analysis: representative images from two independent experiments are shown.
    Figure Legend Snippet: ER stress impairs apoA-I expression. HepG2 cells were incubated with increasing concentrations of thapsigargin (A, B) or 0.1 µM thapsigargin (C) in media containing 10% LPDS for 24 h. ApoA-I mRNA was determined by qRT-PCR and normalized to 18s expression (A). ApoA-I protein expression was analyzed by Western blotting (B) and immunofluorescence analyses (C). Green: ABCA1; blue: DAPI. Bar = 10 µm. apoA-I mRNA and protein are decreased by thapsigargin treatment. qRT-PCR: mean ± SD (n = 3). Western blot and immunofluorescence analysis: representative images from two independent experiments are shown.

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Immunofluorescence

    ABCA1 is regulated independently of LXR during ER stress. A: HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. Gene expression was determined by qRT-PCR and normalized to 18s expression. mRNA expression of ABCA1 was compared with expression of ABCG5 and IDOL, which are established LXR target genes in hepatic cells. ABCA1 is regulated differently compared with ABCG5 and IDOL. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing wild-type or LXR-binding site (DR-4) mutated constructs of the ABCA1 core promoter (−175/+224). After 4 h, cells were treated with 0.1 µM thapsigargin and/or 5 µM of LXR agonist TO901317 in media containing 10% LPDS for another 24 h. Promoter activity was determined using dual luciferase assay. Data show one representative experiment out of three independently performed experiments. Promoter activity was normalized to the activity of the wild-type construct under untreated conditions.
    Figure Legend Snippet: ABCA1 is regulated independently of LXR during ER stress. A: HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. Gene expression was determined by qRT-PCR and normalized to 18s expression. mRNA expression of ABCA1 was compared with expression of ABCG5 and IDOL, which are established LXR target genes in hepatic cells. ABCA1 is regulated differently compared with ABCG5 and IDOL. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing wild-type or LXR-binding site (DR-4) mutated constructs of the ABCA1 core promoter (−175/+224). After 4 h, cells were treated with 0.1 µM thapsigargin and/or 5 µM of LXR agonist TO901317 in media containing 10% LPDS for another 24 h. Promoter activity was determined using dual luciferase assay. Data show one representative experiment out of three independently performed experiments. Promoter activity was normalized to the activity of the wild-type construct under untreated conditions.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Luciferase, Binding Assay, Construct, Activity Assay

    ER stress alters cholesterol distribution but not cholesterol content. HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. A: Lipids were extracted, and free and esterified cholesterol were directly quantified by GC. Data show means ± SD from three experiments. CE, esterified cholesterol; FC, free cholesterol; TC, total cholesterol. B: Free cholesterol distribution was visualized by Filipin staining. Cholesterol distribution is altered by ER stress, although no quantitative alterations in FC and CE exist. Bar = 10 µm. Representative images from three independent experiments are shown.
    Figure Legend Snippet: ER stress alters cholesterol distribution but not cholesterol content. HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. A: Lipids were extracted, and free and esterified cholesterol were directly quantified by GC. Data show means ± SD from three experiments. CE, esterified cholesterol; FC, free cholesterol; TC, total cholesterol. B: Free cholesterol distribution was visualized by Filipin staining. Cholesterol distribution is altered by ER stress, although no quantitative alterations in FC and CE exist. Bar = 10 µm. Representative images from three independent experiments are shown.

    Techniques Used: Staining

    Induction of ER stress by altering calcium homeostasis impairs ABCA1 expression and redistributes ABCA1. HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression was determined by qRT-PCR and normalized to 18s expression. The ER stress markers CHOP, ATF4, GRP78, and ERdJ4 are induced dose dependently, whereas ABCA1 mRNA was reduced (A–E). Western blot analysis showed dose-dependent reduction of ABCA1 protein expression by thapsigargin treatment (F). ABCA1 and CHOP mRNA expression is altered inversely over time after thapsigargin (0.1 µM) treatment (G). Thapsigargin treatment (0.1 µM) alters ABCA1 localization to tubular cellular compartments as visualized by immunofluorescence microscopy (h). Green: ABCA1; blue: DAPI; bar = 5 µm. qRT-PCR: mean ± SD (n = 3). For Western blot and immunofluorescence analyses, representative images from three independent experiments are shown.
    Figure Legend Snippet: Induction of ER stress by altering calcium homeostasis impairs ABCA1 expression and redistributes ABCA1. HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression was determined by qRT-PCR and normalized to 18s expression. The ER stress markers CHOP, ATF4, GRP78, and ERdJ4 are induced dose dependently, whereas ABCA1 mRNA was reduced (A–E). Western blot analysis showed dose-dependent reduction of ABCA1 protein expression by thapsigargin treatment (F). ABCA1 and CHOP mRNA expression is altered inversely over time after thapsigargin (0.1 µM) treatment (G). Thapsigargin treatment (0.1 µM) alters ABCA1 localization to tubular cellular compartments as visualized by immunofluorescence microscopy (h). Green: ABCA1; blue: DAPI; bar = 5 µm. qRT-PCR: mean ± SD (n = 3). For Western blot and immunofluorescence analyses, representative images from three independent experiments are shown.

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Immunofluorescence, Microscopy

    5) Product Images from "MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice"

    Article Title: MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

    Journal: Nature Communications

    doi: 10.1038/ncomms3883

    SREBP-1 is regulated by endogenous changes in miR-33 in vitro . ( a ) RNA expression levels in Srebf2 , miR-33 and Srebf1c in primary hepatocytes cultured in DMEM supplemented with 5% FBS or 5% LPDS with or without statin treatment. Values are the mean±s.e.m. ( n =3 each for Srebf2 and Srebf1c , n =4–6 each for miR-33, * P
    Figure Legend Snippet: SREBP-1 is regulated by endogenous changes in miR-33 in vitro . ( a ) RNA expression levels in Srebf2 , miR-33 and Srebf1c in primary hepatocytes cultured in DMEM supplemented with 5% FBS or 5% LPDS with or without statin treatment. Values are the mean±s.e.m. ( n =3 each for Srebf2 and Srebf1c , n =4–6 each for miR-33, * P

    Techniques Used: In Vitro, RNA Expression, Cell Culture

    6) Product Images from "STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites"

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    Journal: The EMBO Journal

    doi: 10.15252/embj.201695917

    STARD3‐mediated cholesterol accumulation in endosomes does not alter cholesterol homeostasis Total free cholesterol quantification after total lipid extraction in control cells and in cells expressing STARD3 or the lipid binding mutant form of STARD3 (STARD3 MR/ND) ( n = 3). Please note that control HeLa cells treated with MβCD have a significant depletion of total cholesterol ( n = 2). Mean ± SD; ANOVA with Tukey's multiple comparison test. Western blot analysis of SREBP‐2 activation in controls (HeLa, HeLa/empty vector) or STARD3‐overexpressing HeLa cells. Cells were incubated for 2 h in DMEM culture medium supplemented with: 5% FCS (NT); 5% LPDS, 10 μM mevinolin (LPDS); 5% LPDS, 10 μM mevinolin, 1 mM MβCD (MβCD); 5% LPDS, 10 μM mevinolin, 500 μM cholesterol complexed to MβCD (MβCD‐Chol). The proteasome inhibitor MG132 (10 μM) was present in all conditions. P = precursor form of SREBP‐2; C = cleaved form of SREBP‐2. Lower panel: WB quantification in which cleaved SREBP‐2 is expressed as a fraction of total SREBP2 (P+C). Mean ± SD; n = 4 independent experiments; ** P
    Figure Legend Snippet: STARD3‐mediated cholesterol accumulation in endosomes does not alter cholesterol homeostasis Total free cholesterol quantification after total lipid extraction in control cells and in cells expressing STARD3 or the lipid binding mutant form of STARD3 (STARD3 MR/ND) ( n = 3). Please note that control HeLa cells treated with MβCD have a significant depletion of total cholesterol ( n = 2). Mean ± SD; ANOVA with Tukey's multiple comparison test. Western blot analysis of SREBP‐2 activation in controls (HeLa, HeLa/empty vector) or STARD3‐overexpressing HeLa cells. Cells were incubated for 2 h in DMEM culture medium supplemented with: 5% FCS (NT); 5% LPDS, 10 μM mevinolin (LPDS); 5% LPDS, 10 μM mevinolin, 1 mM MβCD (MβCD); 5% LPDS, 10 μM mevinolin, 500 μM cholesterol complexed to MβCD (MβCD‐Chol). The proteasome inhibitor MG132 (10 μM) was present in all conditions. P = precursor form of SREBP‐2; C = cleaved form of SREBP‐2. Lower panel: WB quantification in which cleaved SREBP‐2 is expressed as a fraction of total SREBP2 (P+C). Mean ± SD; n = 4 independent experiments; ** P

    Techniques Used: Expressing, Binding Assay, Mutagenesis, Western Blot, Activation Assay, Plasmid Preparation, Incubation

    The ER is the main source of cholesterol accumulated by STARD3 in endosomes HeLa/Ctrl and HeLa/STARD3 cells were incubated in normal medium (A), LPDS‐containing medium (B) or normal medium with 50 μM mevinolin and 100 μM mevalonate (C), for 48 h. Cholesterol accumulation in endosomes was detected by filipin staining (Cyan Hot) in endosomes identified by the presence of Lamp1 (red) and STARD3 (magenta). Nuclei were stained in blue. Merged image of filipin and STARD3 signals is shown in (d and h). The subpanels on the right are higher magnification (3.5×) images of the area outlined in white (a, e). The filipin and STARD3 merged image is labeled Overlay. Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin in HeLa/Ctrl and HeLa/STARD3 cells incubated or not in LPDS‐containing medium (D) or treated or not with mevinolin and mevalonate (E). Mean ± SD; n = 5 (D) and n = 4 (E) independent experiments; ** P
    Figure Legend Snippet: The ER is the main source of cholesterol accumulated by STARD3 in endosomes HeLa/Ctrl and HeLa/STARD3 cells were incubated in normal medium (A), LPDS‐containing medium (B) or normal medium with 50 μM mevinolin and 100 μM mevalonate (C), for 48 h. Cholesterol accumulation in endosomes was detected by filipin staining (Cyan Hot) in endosomes identified by the presence of Lamp1 (red) and STARD3 (magenta). Nuclei were stained in blue. Merged image of filipin and STARD3 signals is shown in (d and h). The subpanels on the right are higher magnification (3.5×) images of the area outlined in white (a, e). The filipin and STARD3 merged image is labeled Overlay. Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin in HeLa/Ctrl and HeLa/STARD3 cells incubated or not in LPDS‐containing medium (D) or treated or not with mevinolin and mevalonate (E). Mean ± SD; n = 5 (D) and n = 4 (E) independent experiments; ** P

    Techniques Used: Incubation, Staining, Labeling, Fluorescence

    7) Product Images from "Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders"

    Article Title: Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32776-6

    FP-5 fluorescence in cells with abnormal content of cholesterol. ( A ) Cholesterol transport in U-2 OS was inhibited by inhibitor U18666A (1 μg/ml) for 48 h and then cells were labelled with FP-5 (200 nM) for additional 24 h, fixed and stained with filipin (50 μg/ml). Expansion of the region indicated by the white box is shown on the low left side. ( B ) Human fibroblasts carrying mutations in NPC1 cholesterol transporter (clones GB03123E, GB18436) and control normal human fibroblasts (HDFa) were labelled with FP-5 (200 nM) for 6 h and examined. ( C ) Co-localization of FP-5 and filipin staining in mutant cell clones. ( D ) Differential kinetics of FP-5 and TF-Chol lysosomal labelling in NPC-GM18436 fibroblasts. Cells were incubated with FP-5 (200 nM) and TF-Chol (1 μM) for indicated times in medium containing 5% LPDS and imaged live. Scale bar represents 10 μM.
    Figure Legend Snippet: FP-5 fluorescence in cells with abnormal content of cholesterol. ( A ) Cholesterol transport in U-2 OS was inhibited by inhibitor U18666A (1 μg/ml) for 48 h and then cells were labelled with FP-5 (200 nM) for additional 24 h, fixed and stained with filipin (50 μg/ml). Expansion of the region indicated by the white box is shown on the low left side. ( B ) Human fibroblasts carrying mutations in NPC1 cholesterol transporter (clones GB03123E, GB18436) and control normal human fibroblasts (HDFa) were labelled with FP-5 (200 nM) for 6 h and examined. ( C ) Co-localization of FP-5 and filipin staining in mutant cell clones. ( D ) Differential kinetics of FP-5 and TF-Chol lysosomal labelling in NPC-GM18436 fibroblasts. Cells were incubated with FP-5 (200 nM) and TF-Chol (1 μM) for indicated times in medium containing 5% LPDS and imaged live. Scale bar represents 10 μM.

    Techniques Used: Fluorescence, Staining, Mutagenesis, Clone Assay, Incubation

    8) Product Images from "Serum albumin acts as a shuttle to enhance cholesterol efflux from cells [S]"

    Article Title: Serum albumin acts as a shuttle to enhance cholesterol efflux from cells [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M031336

    Synergy obtained from J774 cells when LPDS is used as a shuttle and LDL as a sink. Radiolabeled J774 cells were incubated with increasing concentrations of LPDS alone or in combination with 25 µg/ml LDL, and cholesterol efflux was measured after
    Figure Legend Snippet: Synergy obtained from J774 cells when LPDS is used as a shuttle and LDL as a sink. Radiolabeled J774 cells were incubated with increasing concentrations of LPDS alone or in combination with 25 µg/ml LDL, and cholesterol efflux was measured after

    Techniques Used: Incubation

    Cholesterol efflux from J774 cells when LPDS is used as a shuttle and LDL as a sink. Radiolabeled J774 cells were incubated with increasing concentrations of LPDS alone or in combination with 25 µg/ml LDL. Cholesterol efflux was measured after
    Figure Legend Snippet: Cholesterol efflux from J774 cells when LPDS is used as a shuttle and LDL as a sink. Radiolabeled J774 cells were incubated with increasing concentrations of LPDS alone or in combination with 25 µg/ml LDL. Cholesterol efflux was measured after

    Techniques Used: Incubation

    9) Product Images from "Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders"

    Article Title: Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32776-6

    FP-5 fluorescence in cells with abnormal content of cholesterol. ( A ) Cholesterol transport in U-2 OS was inhibited by inhibitor U18666A (1 μg/ml) for 48 h and then cells were labelled with FP-5 (200 nM) for additional 24 h, fixed and stained with filipin (50 μg/ml). Expansion of the region indicated by the white box is shown on the low left side. ( B ) Human fibroblasts carrying mutations in NPC1 cholesterol transporter (clones GB03123E, GB18436) and control normal human fibroblasts (HDFa) were labelled with FP-5 (200 nM) for 6 h and examined. ( C ) Co-localization of FP-5 and filipin staining in mutant cell clones. ( D ) Differential kinetics of FP-5 and TF-Chol lysosomal labelling in NPC-GM18436 fibroblasts. Cells were incubated with FP-5 (200 nM) and TF-Chol (1 μM) for indicated times in medium containing 5% LPDS and imaged live. Scale bar represents 10 μM.
    Figure Legend Snippet: FP-5 fluorescence in cells with abnormal content of cholesterol. ( A ) Cholesterol transport in U-2 OS was inhibited by inhibitor U18666A (1 μg/ml) for 48 h and then cells were labelled with FP-5 (200 nM) for additional 24 h, fixed and stained with filipin (50 μg/ml). Expansion of the region indicated by the white box is shown on the low left side. ( B ) Human fibroblasts carrying mutations in NPC1 cholesterol transporter (clones GB03123E, GB18436) and control normal human fibroblasts (HDFa) were labelled with FP-5 (200 nM) for 6 h and examined. ( C ) Co-localization of FP-5 and filipin staining in mutant cell clones. ( D ) Differential kinetics of FP-5 and TF-Chol lysosomal labelling in NPC-GM18436 fibroblasts. Cells were incubated with FP-5 (200 nM) and TF-Chol (1 μM) for indicated times in medium containing 5% LPDS and imaged live. Scale bar represents 10 μM.

    Techniques Used: Fluorescence, Staining, Mutagenesis, Clone Assay, Incubation

    10) Product Images from "Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1"

    Article Title: Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI16445

    Degradation of 125 I-labeled LDL by skin fibroblasts, EBV-lymphocytes, and monocyte-derived macrophages from individuals in family 3. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) EBV-lymphocytes, ( b ) skin fibroblasts, and ( c ) monocyte-derived macrophages, from probands 3.1 (filled triangles) and 3.2 (open triangles) in family 3 and from unrelated normolipemic controls (filled circles).
    Figure Legend Snippet: Degradation of 125 I-labeled LDL by skin fibroblasts, EBV-lymphocytes, and monocyte-derived macrophages from individuals in family 3. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) EBV-lymphocytes, ( b ) skin fibroblasts, and ( c ) monocyte-derived macrophages, from probands 3.1 (filled triangles) and 3.2 (open triangles) in family 3 and from unrelated normolipemic controls (filled circles).

    Techniques Used: Labeling, Derivative Assay, Radioactivity, Incubation

    Degradation of 125 I-labeled LDL by cultured skin fibroblasts and EBV- lymphocytes from individuals in family 1. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of trichloroacetic acid–soluble (TCA-soluble), non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) Cultured skin fibroblasts from the proband in family 1 (ARH –/– ), and from a normolipemic control (ARH +/+ ). ( b ) EBV-lymphocytes from the proband in family 1 (ARH –/– ), her heterozygous sibling (ARH +/– ), and a sibling who does not carry the mutant allele (ARH +/+ ).
    Figure Legend Snippet: Degradation of 125 I-labeled LDL by cultured skin fibroblasts and EBV- lymphocytes from individuals in family 1. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of trichloroacetic acid–soluble (TCA-soluble), non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) Cultured skin fibroblasts from the proband in family 1 (ARH –/– ), and from a normolipemic control (ARH +/+ ). ( b ) EBV-lymphocytes from the proband in family 1 (ARH –/– ), her heterozygous sibling (ARH +/– ), and a sibling who does not carry the mutant allele (ARH +/+ ).

    Techniques Used: Labeling, Cell Culture, Radioactivity, Incubation, Mutagenesis

    Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. ( a and b ) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon membranes, and immunoblotted with anti–c-myc (lanes 1–8) or anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with peroxidase-conjugated anti-mouse IgG and chemiluminescence. ( a ) Whole-cell extracts (approximately 50 μg of protein per lane) of Chinese hamster ovary (CHO) cells transiently transfected with pcDNA3-c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retroviral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from affected individual 1.1 (ARH1.1 cells) 1 month after infection with c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes from affected individual 1.1 (lane 5). ( b ) Whole-cell extracts (approximately 50 μg of protein per lane) of EBV-lymphocytes from affected individual 1.1 (lane 6), the same cells 3 months after stable infection with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected cells after preincubation for 16 hours with 0.3 μM trichostatin A (lane 8 and 10). ( c ) Virus-infected (ARH – /c-myc-ARH) and uninfected (ARH – ) EBV-lymphocytes from individual 1.1 were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Data shown are representative of two separate experiments.
    Figure Legend Snippet: Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. ( a and b ) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon membranes, and immunoblotted with anti–c-myc (lanes 1–8) or anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with peroxidase-conjugated anti-mouse IgG and chemiluminescence. ( a ) Whole-cell extracts (approximately 50 μg of protein per lane) of Chinese hamster ovary (CHO) cells transiently transfected with pcDNA3-c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retroviral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from affected individual 1.1 (ARH1.1 cells) 1 month after infection with c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes from affected individual 1.1 (lane 5). ( b ) Whole-cell extracts (approximately 50 μg of protein per lane) of EBV-lymphocytes from affected individual 1.1 (lane 6), the same cells 3 months after stable infection with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected cells after preincubation for 16 hours with 0.3 μM trichostatin A (lane 8 and 10). ( c ) Virus-infected (ARH – /c-myc-ARH) and uninfected (ARH – ) EBV-lymphocytes from individual 1.1 were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Data shown are representative of two separate experiments.

    Techniques Used: Expressing, Activity Assay, Mutagenesis, Transfection, Construct, Infection, Labeling, Radioactivity, Incubation

    11) Product Images from "Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-?24-reductase deficient cell line"

    Article Title: Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-?24-reductase deficient cell line

    Journal: The Biochemical journal

    doi: 10.1042/BJ20081909

    Sterol content and biosynthesis in J774 cells adapted to grow in a cholesterol-free medium. ( A ) Cholesterol and desmosterol content of cells at different times of growth after the complete replacement of RPMI containing 10% FBS with either RPMI containing 10% LPDS or DCCM-1. ( B and C ) HPLC profiles of radioactivity ( B ) and absorbance at 206 nm ( C ) of sterols from cells at day 21 of growth in DCCM-1. DCCM-1 was supplemented with 40 μCi of [ 14 C]acetate for 3 days and the cell extracts containing the nonsaponifiable lipid fraction were analyzed by reverse-phase HPLC and in-line radioactivity counting. The arrows indicate the elution times of desmosterol (des), cholesterol (chol) and ergosterol (erg). d, days; m, months; is, internal standard; nd, not detected. ( D ) Average cholesterol and desmosterol levels in cells maintained in RPMI containing 10% FBS or at days 21 to 32 of growth in DCCM-1. Bars show the mean and whiskers indicate the SEM of data from seven separate cell cultures.
    Figure Legend Snippet: Sterol content and biosynthesis in J774 cells adapted to grow in a cholesterol-free medium. ( A ) Cholesterol and desmosterol content of cells at different times of growth after the complete replacement of RPMI containing 10% FBS with either RPMI containing 10% LPDS or DCCM-1. ( B and C ) HPLC profiles of radioactivity ( B ) and absorbance at 206 nm ( C ) of sterols from cells at day 21 of growth in DCCM-1. DCCM-1 was supplemented with 40 μCi of [ 14 C]acetate for 3 days and the cell extracts containing the nonsaponifiable lipid fraction were analyzed by reverse-phase HPLC and in-line radioactivity counting. The arrows indicate the elution times of desmosterol (des), cholesterol (chol) and ergosterol (erg). d, days; m, months; is, internal standard; nd, not detected. ( D ) Average cholesterol and desmosterol levels in cells maintained in RPMI containing 10% FBS or at days 21 to 32 of growth in DCCM-1. Bars show the mean and whiskers indicate the SEM of data from seven separate cell cultures.

    Techniques Used: High Performance Liquid Chromatography, Radioactivity

    Sterol biosynthesis and Dhcr24 expression in J774 cells. ( A ) [ 14 C]Acetate incorporation into sterols in J774 and HL-60 cells. Cells were incubated in RPMI containing 10% LPDS supplemented with 40 μCi of [ 14 C]acetate for 10 h and the cell extracts containing the nonsaponifiable lipid fraction were analyzed by reverse-phase HPLC and in-line radioactivity counting. The arrows indicate the elution times of desmosterol (des), and cholesterol (chol). nd, not detected. ( B ) Northern blot analysis of RNA of J774 cells and mouse liver. Total RNA was subjected to electrophoresis and blot hybridization with digoxygenin-labelled cDNA probes for mouse Dhcr24 and mouse Gapdh . ( C ) RT-PCR analysis of RNA of J774 cells and mouse liver for detection of Dhcr24 and Gapdh . ( D ) RT-PCR analysis of RNA of J774 cells and mouse liver for detection of Dhcr24 ) primers (filled and empty arrowheads, respectively) used for the amplifications and the resulting cDNA fragments (horizontal bars). stand., standard; bp, base pairs.
    Figure Legend Snippet: Sterol biosynthesis and Dhcr24 expression in J774 cells. ( A ) [ 14 C]Acetate incorporation into sterols in J774 and HL-60 cells. Cells were incubated in RPMI containing 10% LPDS supplemented with 40 μCi of [ 14 C]acetate for 10 h and the cell extracts containing the nonsaponifiable lipid fraction were analyzed by reverse-phase HPLC and in-line radioactivity counting. The arrows indicate the elution times of desmosterol (des), and cholesterol (chol). nd, not detected. ( B ) Northern blot analysis of RNA of J774 cells and mouse liver. Total RNA was subjected to electrophoresis and blot hybridization with digoxygenin-labelled cDNA probes for mouse Dhcr24 and mouse Gapdh . ( C ) RT-PCR analysis of RNA of J774 cells and mouse liver for detection of Dhcr24 and Gapdh . ( D ) RT-PCR analysis of RNA of J774 cells and mouse liver for detection of Dhcr24 ) primers (filled and empty arrowheads, respectively) used for the amplifications and the resulting cDNA fragments (horizontal bars). stand., standard; bp, base pairs.

    Techniques Used: Expressing, Incubation, High Performance Liquid Chromatography, Radioactivity, Northern Blot, Electrophoresis, Hybridization, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1"

    Article Title: Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI16445

    Degradation of 125 I-labeled LDL by skin fibroblasts, EBV-lymphocytes, and monocyte-derived macrophages from individuals in family 3. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) EBV-lymphocytes, ( b ) skin fibroblasts, and ( c ) monocyte-derived macrophages, from probands 3.1 (filled triangles) and 3.2 (open triangles) in family 3 and from unrelated normolipemic controls (filled circles).
    Figure Legend Snippet: Degradation of 125 I-labeled LDL by skin fibroblasts, EBV-lymphocytes, and monocyte-derived macrophages from individuals in family 3. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) EBV-lymphocytes, ( b ) skin fibroblasts, and ( c ) monocyte-derived macrophages, from probands 3.1 (filled triangles) and 3.2 (open triangles) in family 3 and from unrelated normolipemic controls (filled circles).

    Techniques Used: Labeling, Derivative Assay, Radioactivity, Incubation

    Degradation of 125 I-labeled LDL by cultured skin fibroblasts and EBV- lymphocytes from individuals in family 1. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of trichloroacetic acid–soluble (TCA-soluble), non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) Cultured skin fibroblasts from the proband in family 1 (ARH –/– ), and from a normolipemic control (ARH +/+ ). ( b ) EBV-lymphocytes from the proband in family 1 (ARH –/– ), her heterozygous sibling (ARH +/– ), and a sibling who does not carry the mutant allele (ARH +/+ ).
    Figure Legend Snippet: Degradation of 125 I-labeled LDL by cultured skin fibroblasts and EBV- lymphocytes from individuals in family 1. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of trichloroacetic acid–soluble (TCA-soluble), non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Nonsaturable degradation of LDL by normal cells was always less than 5% of the total. Data shown are representative of at least two separate experiments. ( a ) Cultured skin fibroblasts from the proband in family 1 (ARH –/– ), and from a normolipemic control (ARH +/+ ). ( b ) EBV-lymphocytes from the proband in family 1 (ARH –/– ), her heterozygous sibling (ARH +/– ), and a sibling who does not carry the mutant allele (ARH +/+ ).

    Techniques Used: Labeling, Cell Culture, Radioactivity, Incubation, Mutagenesis

    Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. ( a and b ) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon membranes, and immunoblotted with anti–c-myc (lanes 1–8) or anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with peroxidase-conjugated anti-mouse IgG and chemiluminescence. ( a ) Whole-cell extracts (approximately 50 μg of protein per lane) of Chinese hamster ovary (CHO) cells transiently transfected with pcDNA3-c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retroviral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from affected individual 1.1 (ARH1.1 cells) 1 month after infection with c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes from affected individual 1.1 (lane 5). ( b ) Whole-cell extracts (approximately 50 μg of protein per lane) of EBV-lymphocytes from affected individual 1.1 (lane 6), the same cells 3 months after stable infection with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected cells after preincubation for 16 hours with 0.3 μM trichostatin A (lane 8 and 10). ( c ) Virus-infected (ARH – /c-myc-ARH) and uninfected (ARH – ) EBV-lymphocytes from individual 1.1 were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Data shown are representative of two separate experiments.
    Figure Legend Snippet: Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. ( a and b ) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon membranes, and immunoblotted with anti–c-myc (lanes 1–8) or anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with peroxidase-conjugated anti-mouse IgG and chemiluminescence. ( a ) Whole-cell extracts (approximately 50 μg of protein per lane) of Chinese hamster ovary (CHO) cells transiently transfected with pcDNA3-c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retroviral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from affected individual 1.1 (ARH1.1 cells) 1 month after infection with c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes from affected individual 1.1 (lane 5). ( b ) Whole-cell extracts (approximately 50 μg of protein per lane) of EBV-lymphocytes from affected individual 1.1 (lane 6), the same cells 3 months after stable infection with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected cells after preincubation for 16 hours with 0.3 μM trichostatin A (lane 8 and 10). ( c ) Virus-infected (ARH – /c-myc-ARH) and uninfected (ARH – ) EBV-lymphocytes from individual 1.1 were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125 I-labeled LDL. Saturable degradation of LDL was determined as the difference in the amount of TCA-soluble, non-iodide radioactivity in the medium of cells incubated in the presence and absence of an excess of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes. Data shown are representative of two separate experiments.

    Techniques Used: Expressing, Activity Assay, Mutagenesis, Transfection, Construct, Infection, Labeling, Radioactivity, Incubation

    13) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1091/mbc.E06-10-0924

    (A) MβCD and LPDS treatment alter rab11 distribution and the internalization of fluorescent SM in confluent cells. (A) Cells were treated with MβCD or LPDS as described in Materials and Methods. Cells were then fixed, labeled with anti-rab11
    Figure Legend Snippet: (A) MβCD and LPDS treatment alter rab11 distribution and the internalization of fluorescent SM in confluent cells. (A) Cells were treated with MβCD or LPDS as described in Materials and Methods. Cells were then fixed, labeled with anti-rab11

    Techniques Used: Labeling

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    Planar Chromatography:

    Article Title: Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles
    Article Snippet: Bound antibodies were detected with goat anti-mouse antibody conjugated to horseradish peroxidase (Promega, Lyon, France) and enhanced chemiluminescence. .. Human hepatoma cell lines PLC/PFR/5 (PLC) and HepG2 were cultured in DMEM (Gibco/BRL) supplemented with 10% fetal calf serum (FCS) (Biowhittaker, Emerainville, France) or 10% lipoprotein-deficient FCS (LPDS) (Sigma), 2 mM HEPES (Gibco/BRL), 1% nonessential amino acids (Gibco/BRL), and 50 IU of penicillin-streptomycin (Gibco/BRL)/ml at 37°C in a 5% CO2 atmosphere; cultures were split twice a week. .. HepG2 clone N3 stably expressing the HCV core protein was from T. Miyamura ( ).

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    Millipore lpds
    Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in <t>LPDS</t> followed by <t>transfection</t> with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .
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    Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in LPDS followed by transfection with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .

    Journal: The Journal of Neuroscience

    Article Title: Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction

    doi: 10.1523/JNEUROSCI.0686-09.2009

    Figure Lengend Snippet: Reconstituting cholesterol-dependent myelin protein trafficking in heterologous cells. A , Structure of the transfected fusion proteins wild-type (wt) P0–CFP and YFP–MAG. For live staining experiments determining surface localization (as shown in D ), antibodies directed against the extracellular domain of P0 (anti-P0) and against the fluorescent protein tag (anti-FP) were used. B , Quantification of surface localization of P0–CFP and YFP–MAG in experiments as shown in D and E . In the presence of cholesterol, the relative surface localization of wild-type P0 (normalized to relative surface localization of MAG in the same cell) was 1.3 ± 0.3 but only 0.4 ± 0.1 without added cholesterol. In the presence of cholesterol, relative surface localization of mutant (mut) P0 to MAG in the same cell was 1.4 ± 0.1 and 1.0 ± 0.1 without added cholesterol. C , Structure of transfected fusion proteins mutated P0–CFP (mutant P0–CFP, Y148S R151L) and YFP–MAG. Surface localization was determined by life stain experiments using anti-P0 antibodies and against the fluorescent protein tag (anti-FP). D , SQS null fibroblasts that were starved of cholesterol for 24 h in LPDS followed by transfection with both wild-type P0–CFP and MAG–YFP and cultivation for 24 h in LPDS with 10 μg/ml cholesterol (chol) or without cholesterol (w/o chol). Surface localization of transfected proteins was visualized as described in A . Merged images show surface immunostain in red with the corresponding fluorescent protein in green. Scale bar, 10 μm. E , SQS null fibroblasts were transfected with both mutated P0–CFP and MAG–YFP as described in D .

    Article Snippet: For transfection experiments, fibroblasts were starved in 2% LPDS (Sigma) for 24 h and transfected with tagged versions of myelin-associated glycoprotein (MAG) and P0 in LPDS with or without 10 μg/ml cholesterol using Lipofectamine2000 (Invitrogen).

    Techniques: Transfection, Staining, Mutagenesis

    Identification of potential cholesterol-binding motifs in RHBDL4. A, human RHBDL4 protein sequence is displayed with the six transmembrane regions highlighted in gray (determined by homology with GlpG). Furthermore, potential CRAC motifs and the mirror version ( CARC ) are highlighted. B, ). Serine 144 and histidine 195 are displayed as sticks and form the active center. Tyr-106 in TMS3 and Tyr-205 in TMS6 are highlighted. C, comparison of RHBDL4 sequences from a total of 72 species (11 examples displayed here) show a high degree of conservation across species for the CRAC motif in TMS6. D, point mutations in RHBDL4 increase protease activity. Tyr-106 and Tyr-205 were mutated to alanine. RHBDL4 mutants were transfected into HEK 293T cells, and the generation of endogenous APP CTFs was determined by Western blot analysis. Representative Western blotting of 7–9 independent experiments is displayed. F, analysis of APP mRNA levels upon transfection of RHBDL4 WT, Y106A, Y205A, and Y106A/Y205A. No significant differences for APP mRNA were detected. GAPDH and β-actin served as reference genes. Data are displayed as mean expression ± S.E., n = 3. One-way analysis of variance followed by Dunnett's post hoc comparison with WT as control was performed. G, RHBDL4 mutants are not responsive to LPDS treatment. 12 h post-transfection, HEK 293T cells were treated with either 10% FCS or LPDS in DMEM for 24 h. A representative Western blotting of six independent experiments is shown. E and H, APP CTFs or APP full length were quantified, and results were normalized to β-actin and then either compared with WT ( D ) or FCS treatment ( F ). D, WT was set to 1 and is indicated by the blue dashed line , and F, FCS-treated samples were set to 1. Mean ± S.E. are displayed, n = 6–9 as stated above, p values for Holm-Bonferroni corrected one sample t test are reported. I, pulldown of RHBDL4 with biotinylated cholesterol. RHBDL4 WT and mutants were transfected, and membrane preparations were incubated with alkyne-cholesterol. After clicking biotin-azide to cholesterol, a neutravidin pulldown was performed. Total cell lysates were collected before the click chemistry was performed, and the Western blotting shows expression of RHBDL4 WT and mutants. Representative Western blots of 3–4 independent experiments are shown. D, F, and I, detection of APP full length and endogenous CTFs with Y188, RHBDL4 with anti-Myc antibody, and β-actin as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Membrane cholesterol as regulator of human rhomboid protease RHBDL4

    doi: 10.1074/jbc.RA118.002640

    Figure Lengend Snippet: Identification of potential cholesterol-binding motifs in RHBDL4. A, human RHBDL4 protein sequence is displayed with the six transmembrane regions highlighted in gray (determined by homology with GlpG). Furthermore, potential CRAC motifs and the mirror version ( CARC ) are highlighted. B, ). Serine 144 and histidine 195 are displayed as sticks and form the active center. Tyr-106 in TMS3 and Tyr-205 in TMS6 are highlighted. C, comparison of RHBDL4 sequences from a total of 72 species (11 examples displayed here) show a high degree of conservation across species for the CRAC motif in TMS6. D, point mutations in RHBDL4 increase protease activity. Tyr-106 and Tyr-205 were mutated to alanine. RHBDL4 mutants were transfected into HEK 293T cells, and the generation of endogenous APP CTFs was determined by Western blot analysis. Representative Western blotting of 7–9 independent experiments is displayed. F, analysis of APP mRNA levels upon transfection of RHBDL4 WT, Y106A, Y205A, and Y106A/Y205A. No significant differences for APP mRNA were detected. GAPDH and β-actin served as reference genes. Data are displayed as mean expression ± S.E., n = 3. One-way analysis of variance followed by Dunnett's post hoc comparison with WT as control was performed. G, RHBDL4 mutants are not responsive to LPDS treatment. 12 h post-transfection, HEK 293T cells were treated with either 10% FCS or LPDS in DMEM for 24 h. A representative Western blotting of six independent experiments is shown. E and H, APP CTFs or APP full length were quantified, and results were normalized to β-actin and then either compared with WT ( D ) or FCS treatment ( F ). D, WT was set to 1 and is indicated by the blue dashed line , and F, FCS-treated samples were set to 1. Mean ± S.E. are displayed, n = 6–9 as stated above, p values for Holm-Bonferroni corrected one sample t test are reported. I, pulldown of RHBDL4 with biotinylated cholesterol. RHBDL4 WT and mutants were transfected, and membrane preparations were incubated with alkyne-cholesterol. After clicking biotin-azide to cholesterol, a neutravidin pulldown was performed. Total cell lysates were collected before the click chemistry was performed, and the Western blotting shows expression of RHBDL4 WT and mutants. Representative Western blots of 3–4 independent experiments are shown. D, F, and I, detection of APP full length and endogenous CTFs with Y188, RHBDL4 with anti-Myc antibody, and β-actin as a loading control.

    Article Snippet: The cell culture medium was replaced 36 h after transfection with DMEM containing 10% LPDS instead of FCS and supplemented with either 5 μ m simvastatin (stock in DMSO, Sigma) alone or co-treated with 0.25 m m mevalonate (stock in water, Sigma), 40 μg/ml LDL (stock in water, Millipore), or 5 or 10 μ m PF-429242 (stock in DMSO, Sigma) for 24 or 48 h, respectively.

    Techniques: Binding Assay, Sequencing, Activity Assay, Transfection, Western Blot, Expressing, Incubation

    Inhibition of LDs biosynthesis in HBV-producing cells. (A) HepG2 2.2.15 cells were grown for 24 h in complete medium containing either FBS or LPDS. LDs were stained with Bodipy 493/503 and visualized using a Nikon E600 fluorescence microscope. (B) HepG2

    Journal: Journal of Virology

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    doi: 10.1128/JVI.05423-11

    Figure Lengend Snippet: Inhibition of LDs biosynthesis in HBV-producing cells. (A) HepG2 2.2.15 cells were grown for 24 h in complete medium containing either FBS or LPDS. LDs were stained with Bodipy 493/503 and visualized using a Nikon E600 fluorescence microscope. (B) HepG2

    Article Snippet: In cholesterol depletion experiments, FBS was replaced by LPDS (Sigma-Aldrich).

    Techniques: Inhibition, Staining, Fluorescence, Microscopy

    HBV secretion from LPDS-treated cells. HepG2 2.2.15 cells were grown for 24 h in complete medium supplemented with 10% FBS, LPDS, or a mixture of the two. Where indicated, Lova or Cpz was added as a control. (A) The amount of HBV DNA secreted by an equal

    Journal: Journal of Virology

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    doi: 10.1128/JVI.05423-11

    Figure Lengend Snippet: HBV secretion from LPDS-treated cells. HepG2 2.2.15 cells were grown for 24 h in complete medium supplemented with 10% FBS, LPDS, or a mixture of the two. Where indicated, Lova or Cpz was added as a control. (A) The amount of HBV DNA secreted by an equal

    Article Snippet: In cholesterol depletion experiments, FBS was replaced by LPDS (Sigma-Aldrich).

    Techniques:

    Evaluation of the L protein topology in purified microsomes. Cells were grown in complete medium supplemented with 10% FBS or LPDS for 48 h. Following treatment, microsomes were purified from an equal number of cells, split into three aliquots, and treated

    Journal: Journal of Virology

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    doi: 10.1128/JVI.05423-11

    Figure Lengend Snippet: Evaluation of the L protein topology in purified microsomes. Cells were grown in complete medium supplemented with 10% FBS or LPDS for 48 h. Following treatment, microsomes were purified from an equal number of cells, split into three aliquots, and treated

    Article Snippet: In cholesterol depletion experiments, FBS was replaced by LPDS (Sigma-Aldrich).

    Techniques: Purification

    LPDS treatment results in rapid decrease of cholesterol levels in HepG2 2.2.15 cells. Cells were grown for 24 h (A and C) or the times indicated (B) in complete medium supplemented with 10% FBS (considered untreated), 10% LPDS, or a mixture of the two.

    Journal: Journal of Virology

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    doi: 10.1128/JVI.05423-11

    Figure Lengend Snippet: LPDS treatment results in rapid decrease of cholesterol levels in HepG2 2.2.15 cells. Cells were grown for 24 h (A and C) or the times indicated (B) in complete medium supplemented with 10% FBS (considered untreated), 10% LPDS, or a mixture of the two.

    Article Snippet: In cholesterol depletion experiments, FBS was replaced by LPDS (Sigma-Aldrich).

    Techniques:

    HBV replication and viral envelope protein biosynthesis in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for 24, 48, and 72 h. When indicated, 3TC treatment was included as a control of replication

    Journal: Journal of Virology

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    doi: 10.1128/JVI.05423-11

    Figure Lengend Snippet: HBV replication and viral envelope protein biosynthesis in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for 24, 48, and 72 h. When indicated, 3TC treatment was included as a control of replication

    Article Snippet: In cholesterol depletion experiments, FBS was replaced by LPDS (Sigma-Aldrich).

    Techniques:

    Quantification of intracellular nucleocapsids and enveloped virions in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for the times indicated. Cell lysate levels were normalized to the total protein

    Journal: Journal of Virology

    Article Title: Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein ▿

    doi: 10.1128/JVI.05423-11

    Figure Lengend Snippet: Quantification of intracellular nucleocapsids and enveloped virions in LPDS-treated cells. HepG2 2.2.15 cells were grown in complete medium supplemented with 10% FBS or LPDS for the times indicated. Cell lysate levels were normalized to the total protein

    Article Snippet: In cholesterol depletion experiments, FBS was replaced by LPDS (Sigma-Aldrich).

    Techniques:

    Endocytosis pathway for purified LVP. (A) A total of 200,000 copies of HCV RNA from purified LVP were incubated for 3 h with 50,000 HepG2 cells grown for 24 h in LPDS or FCS. Internalized HCV RNA was calculated after suramin treatment. (B) A total of 500,000 copies of HCV RNA from purified LVP were incubated with 20,000 N1 or FH fibroblasts for 3 h before suramin treatment and quantitation of internalized HCV RNA. Error bars indicated standard deviations.

    Journal: Journal of Virology

    Article Title: Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

    doi: 10.1128/JVI.76.14.6919-6928.2002

    Figure Lengend Snippet: Endocytosis pathway for purified LVP. (A) A total of 200,000 copies of HCV RNA from purified LVP were incubated for 3 h with 50,000 HepG2 cells grown for 24 h in LPDS or FCS. Internalized HCV RNA was calculated after suramin treatment. (B) A total of 500,000 copies of HCV RNA from purified LVP were incubated with 20,000 N1 or FH fibroblasts for 3 h before suramin treatment and quantitation of internalized HCV RNA. Error bars indicated standard deviations.

    Article Snippet: Human hepatoma cell lines PLC/PFR/5 (PLC) and HepG2 were cultured in DMEM (Gibco/BRL) supplemented with 10% fetal calf serum (FCS) (Biowhittaker, Emerainville, France) or 10% lipoprotein-deficient FCS (LPDS) (Sigma), 2 mM HEPES (Gibco/BRL), 1% nonessential amino acids (Gibco/BRL), and 50 IU of penicillin-streptomycin (Gibco/BRL)/ml at 37°C in a 5% CO2 atmosphere; cultures were split twice a week.

    Techniques: Purification, Incubation, Quantitation Assay

    Characterization of LVP binding to the LDL receptor. Purified LVP (6.6 × 10 6 HCV RNA copies) were incubated for 1 h with 200 μg of purified human IgG/ml. A total of 300,000 HCV RNA copies were diluted in FCS-free medium supplemented with 0.2% BSA and 50 μg of each anti-ApoB or anti-ApoE monoclonal antibody/ml alone or together. The samples were allowed to stand at room temperature for 1 h with rocking. The samples were then incubated for 45 min on 40,000 HepG2 cells that had been grown for 24 h in medium supplemented with LPDS. Quantitation of cell-associated HCV RNA was then performed. Monoclonal antibodies were 4G3 (anti-ApoB), 5E11 (anti-ApoB), and 1D7 (anti-ApoE). The P value for a comparison of results obtained with the control and the pool of antibodies was

    Journal: Journal of Virology

    Article Title: Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

    doi: 10.1128/JVI.76.14.6919-6928.2002

    Figure Lengend Snippet: Characterization of LVP binding to the LDL receptor. Purified LVP (6.6 × 10 6 HCV RNA copies) were incubated for 1 h with 200 μg of purified human IgG/ml. A total of 300,000 HCV RNA copies were diluted in FCS-free medium supplemented with 0.2% BSA and 50 μg of each anti-ApoB or anti-ApoE monoclonal antibody/ml alone or together. The samples were allowed to stand at room temperature for 1 h with rocking. The samples were then incubated for 45 min on 40,000 HepG2 cells that had been grown for 24 h in medium supplemented with LPDS. Quantitation of cell-associated HCV RNA was then performed. Monoclonal antibodies were 4G3 (anti-ApoB), 5E11 (anti-ApoB), and 1D7 (anti-ApoE). The P value for a comparison of results obtained with the control and the pool of antibodies was

    Article Snippet: Human hepatoma cell lines PLC/PFR/5 (PLC) and HepG2 were cultured in DMEM (Gibco/BRL) supplemented with 10% fetal calf serum (FCS) (Biowhittaker, Emerainville, France) or 10% lipoprotein-deficient FCS (LPDS) (Sigma), 2 mM HEPES (Gibco/BRL), 1% nonessential amino acids (Gibco/BRL), and 50 IU of penicillin-streptomycin (Gibco/BRL)/ml at 37°C in a 5% CO2 atmosphere; cultures were split twice a week.

    Techniques: Binding Assay, Purification, Incubation, Quantitation Assay