u18666a  (Millipore)


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    Name:
    U 18666A
    Description:

    Catalog Number:
    u3633
    Price:
    None
    Applications:
    U 18666A has been used in fibroblast controls to study the mechanism of increase in autophagy in Niemann-Pick Disease Type C (NPC) cells.
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    Structured Review

    Millipore u18666a
    U 18666A

    https://www.bioz.com/result/u18666a/product/Millipore
    Average 99 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    u18666a - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action"

    Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095598

    Pharmacological and genetic inhibition of NPC1 reduced insulin signalling in 3T3-L1 adipocytes. NPC1 was inhibited in 3T3-L1 adipocytes through treatment with U18666a or through siRNA-mediated knockdown of NPC1. ( a ) Subcellular localisation of cholesterol in 3T3-L1 adipocytes after treatment with U18666a for indicated times was visualised by Filipin staining (scale bar = 10 µm), bottom panel. Corresponding bright field images are provided (top panel). Images are representative of n ≥300 cells viewed per condition over 3 experiments. ( b ) Total membrane cholesterol levels within the total membrane fraction. Data are mean ± S.E.M, n = 3 independent experiments, one-way ANOVA. ( c ) Insulin signalling was assessed by immunoblotting total cell lysates of unstimulated and insulin-stimulated (100 nM, 20 min) 3T3-L1 adipocytes for phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3. ( d ) Total protein expression of key proteins in the insulin pathway determined by immunoblotting for IRS-1, IRβ and Akt. ( e ) Subcellular localisation of cholesterol after treatment with scrambled or NPC1 siRNA was visualised by Filipin staining (scale bar = 10 µm). Images are representative of n≥300 cells viewed per condition over 3 experiments. ( f ) The extent of siRNA mediated knockdown of NPC1 and the effect of knockdown on expression of proximal insulin signalling components was measured by probing for NPC1, IRS-1, IRβ and Akt. ( g ) The effect of NPC1 knock-down on insulin signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3 antibodies. ( h ) The levels of insulin-stimulated pS473 and pT308 of Akt in cells NPC1 knockdown cells was quantified relative to control cells. Data are mean ± S.E.M, n = 3 independent experiments, two-sample t-test. ( i ) 3T3-L1 adipocytes overexpressing PDGF receptor were treated with U18666a for 72 hr prior to assessment of insulin and PDGF-mediated (20 ng/mL, 20 min) signalling. Signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt and 14-3-3 antibodies. All data are representative of at least n = 3 independent experiments, significance calculated compared to control cells, n.s = non-significant, *, = p
    Figure Legend Snippet: Pharmacological and genetic inhibition of NPC1 reduced insulin signalling in 3T3-L1 adipocytes. NPC1 was inhibited in 3T3-L1 adipocytes through treatment with U18666a or through siRNA-mediated knockdown of NPC1. ( a ) Subcellular localisation of cholesterol in 3T3-L1 adipocytes after treatment with U18666a for indicated times was visualised by Filipin staining (scale bar = 10 µm), bottom panel. Corresponding bright field images are provided (top panel). Images are representative of n ≥300 cells viewed per condition over 3 experiments. ( b ) Total membrane cholesterol levels within the total membrane fraction. Data are mean ± S.E.M, n = 3 independent experiments, one-way ANOVA. ( c ) Insulin signalling was assessed by immunoblotting total cell lysates of unstimulated and insulin-stimulated (100 nM, 20 min) 3T3-L1 adipocytes for phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3. ( d ) Total protein expression of key proteins in the insulin pathway determined by immunoblotting for IRS-1, IRβ and Akt. ( e ) Subcellular localisation of cholesterol after treatment with scrambled or NPC1 siRNA was visualised by Filipin staining (scale bar = 10 µm). Images are representative of n≥300 cells viewed per condition over 3 experiments. ( f ) The extent of siRNA mediated knockdown of NPC1 and the effect of knockdown on expression of proximal insulin signalling components was measured by probing for NPC1, IRS-1, IRβ and Akt. ( g ) The effect of NPC1 knock-down on insulin signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3 antibodies. ( h ) The levels of insulin-stimulated pS473 and pT308 of Akt in cells NPC1 knockdown cells was quantified relative to control cells. Data are mean ± S.E.M, n = 3 independent experiments, two-sample t-test. ( i ) 3T3-L1 adipocytes overexpressing PDGF receptor were treated with U18666a for 72 hr prior to assessment of insulin and PDGF-mediated (20 ng/mL, 20 min) signalling. Signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt and 14-3-3 antibodies. All data are representative of at least n = 3 independent experiments, significance calculated compared to control cells, n.s = non-significant, *, = p

    Techniques Used: Inhibition, Staining, Expressing

    Inhibition of NPC1 reduced GLUT4 expression via lower LXR activity. ( a ) Total protein levels of IRAP, Transferrin, GLUT1 and GLUT4 were determined by immunoblotting of whole cell lysates from cells with U18666a for indicated times. ( b ) GLUT1, GLUT4, IRAP, and TFR mRNA levels in 3T3-L1 adipocytes treated with U18666a for 72 h were determined by qPCR and presented relative to expression in control cells. Data are mean ±S.E.M, n = 3–5 independent experiments. ( c ) NPC1, GLUT1, GLUT4, IRAP and TFR mRNA levels in 3T3-L1 adipocytes treated with NPC1 siRNA were determined by qPCR and presented relative to expression in control cells. Data are mean ±S.E.M, n = 3 independent experiments. ( d ) Total protein levels of NPC1 and GLUT4 were determined by immunoblotting of whole cell lysates from control and NPC1 knockdown cells. ( e ) mRNA levels of ABCA1 , ABCG1 and APO-E and GLUT4 were determined by qPCR. Control and U18666a (72 h) pre-treated adipocytes were treated with the LXR agonist GW3965 (10 µM) for 16 h. Data are mean ±S.E.M and expressed relative to mRNA levels for each gene in control cells. All data are n = 3–5 independent experiments . ( f ) Percentage change in GLUT4 mRNA levels following GW3095 addition was calculated from (f). ( g ) Total GLUT4 protein levels were determined by immunoblotting of whole cell lysates from control cells and cells treated with U18666a and/or GW3095 as indicated. ( h ) ABCA1, ABCG1 and APOE mRNA levels in 3T3-L1 adipocytes treated with NPC1 siRNA were determined by qPCR and presented relative to expression in control cells. Data are mean ±S.E.M, n = 3 independent experiments. Significance calculated by two sample t-test (b,c,f,h) or two-way ANOVA (e) and indicated p values are compared to control cells n.s = non-significant, * = p
    Figure Legend Snippet: Inhibition of NPC1 reduced GLUT4 expression via lower LXR activity. ( a ) Total protein levels of IRAP, Transferrin, GLUT1 and GLUT4 were determined by immunoblotting of whole cell lysates from cells with U18666a for indicated times. ( b ) GLUT1, GLUT4, IRAP, and TFR mRNA levels in 3T3-L1 adipocytes treated with U18666a for 72 h were determined by qPCR and presented relative to expression in control cells. Data are mean ±S.E.M, n = 3–5 independent experiments. ( c ) NPC1, GLUT1, GLUT4, IRAP and TFR mRNA levels in 3T3-L1 adipocytes treated with NPC1 siRNA were determined by qPCR and presented relative to expression in control cells. Data are mean ±S.E.M, n = 3 independent experiments. ( d ) Total protein levels of NPC1 and GLUT4 were determined by immunoblotting of whole cell lysates from control and NPC1 knockdown cells. ( e ) mRNA levels of ABCA1 , ABCG1 and APO-E and GLUT4 were determined by qPCR. Control and U18666a (72 h) pre-treated adipocytes were treated with the LXR agonist GW3965 (10 µM) for 16 h. Data are mean ±S.E.M and expressed relative to mRNA levels for each gene in control cells. All data are n = 3–5 independent experiments . ( f ) Percentage change in GLUT4 mRNA levels following GW3095 addition was calculated from (f). ( g ) Total GLUT4 protein levels were determined by immunoblotting of whole cell lysates from control cells and cells treated with U18666a and/or GW3095 as indicated. ( h ) ABCA1, ABCG1 and APOE mRNA levels in 3T3-L1 adipocytes treated with NPC1 siRNA were determined by qPCR and presented relative to expression in control cells. Data are mean ±S.E.M, n = 3 independent experiments. Significance calculated by two sample t-test (b,c,f,h) or two-way ANOVA (e) and indicated p values are compared to control cells n.s = non-significant, * = p

    Techniques Used: Inhibition, Expressing, Activity Assay, Real-time Polymerase Chain Reaction

    Inhibition of NPC1 repressed insulin-stimulated glucose uptake. ( a ) 3T3-L1 adipocytes were treated with U18666a for indicated periods of time before radiolabelled 2-deoxy-D-glucose uptake assays were performed on basal and insulin-stimulated cells. Data are mean ±S.E.M, n = 4 independent experiments. ( b ) 3T3-L1 adipocytes electroporated with scrambled (Scr) or siRNA directed to NPC1 (NPC1) were subjected to radiolabelled 2-deoxy-D-glucose uptake assays under basal and insulin-stimulated conditions. Insulin doses as indicated. Data are mean ±S.E.M, n = 3 independent experiments. ( c ) Plasma membrane (PM) levels of IRAP, GLUT4 and Caveolin 1 in unstimulated and insulin-stimulated (100 nM, 20 min) adipocytes were determined by immunoblotting. ( d ) PM levels of HA-GLUT4 was measured using a fluorescence based assay in 3T3-L1 adipocytes overexpressing HA-GLUT4 treated with U18666a for indicated periods of time. Data are mean ±S.E.M, n = 3 independent experiments. Significance calculated by two-way ANOVA. Comparisons to unstimulated cells (a, b, d) indicated by n.s = non-significant, * = p
    Figure Legend Snippet: Inhibition of NPC1 repressed insulin-stimulated glucose uptake. ( a ) 3T3-L1 adipocytes were treated with U18666a for indicated periods of time before radiolabelled 2-deoxy-D-glucose uptake assays were performed on basal and insulin-stimulated cells. Data are mean ±S.E.M, n = 4 independent experiments. ( b ) 3T3-L1 adipocytes electroporated with scrambled (Scr) or siRNA directed to NPC1 (NPC1) were subjected to radiolabelled 2-deoxy-D-glucose uptake assays under basal and insulin-stimulated conditions. Insulin doses as indicated. Data are mean ±S.E.M, n = 3 independent experiments. ( c ) Plasma membrane (PM) levels of IRAP, GLUT4 and Caveolin 1 in unstimulated and insulin-stimulated (100 nM, 20 min) adipocytes were determined by immunoblotting. ( d ) PM levels of HA-GLUT4 was measured using a fluorescence based assay in 3T3-L1 adipocytes overexpressing HA-GLUT4 treated with U18666a for indicated periods of time. Data are mean ±S.E.M, n = 3 independent experiments. Significance calculated by two-way ANOVA. Comparisons to unstimulated cells (a, b, d) indicated by n.s = non-significant, * = p

    Techniques Used: Inhibition, Fluorescence

    2) Product Images from "Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease"

    Article Title: Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147208

    Total Cholesterol and GSLs accumulate in macrophages exposed to U18666A. (A) total cholesterol, (B) ceramide, (C) sphingosine, (D) glucosylceramide (GlcCer), (E) dihydro-ceramide (Dihydro-Cer), and (F) lactosylceramide (LacCer) levels in RAW264.7 murine macrophages exposed to 1, 5 and 10 μM of U18666A for 24 h. Data ( n = 3, mean ± S.E.M.) were analysed using an unpaired t-test. * P
    Figure Legend Snippet: Total Cholesterol and GSLs accumulate in macrophages exposed to U18666A. (A) total cholesterol, (B) ceramide, (C) sphingosine, (D) glucosylceramide (GlcCer), (E) dihydro-ceramide (Dihydro-Cer), and (F) lactosylceramide (LacCer) levels in RAW264.7 murine macrophages exposed to 1, 5 and 10 μM of U18666A for 24 h. Data ( n = 3, mean ± S.E.M.) were analysed using an unpaired t-test. * P

    Techniques Used:

    GSL synthesis inhibition abrogates Gpnmb induction. (A) Total cholesterol, ceramide, glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels of RAW264.7 murine macrophages exposed to 10 μM U18666A and/or 250 μM NB-DNJ for 20 h. (B) Gpnmb, Ccl3 and Cathepsin D (CtsD) gene expression, and sGpnmb levels of RAW264.7 cells exposed to 10 μM U18666A and/or 250 μM NB-DNJ for 20 h. Data ( n = 3 mean ± S.E.M.) were analysed using an unpaired t-test. * P
    Figure Legend Snippet: GSL synthesis inhibition abrogates Gpnmb induction. (A) Total cholesterol, ceramide, glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels of RAW264.7 murine macrophages exposed to 10 μM U18666A and/or 250 μM NB-DNJ for 20 h. (B) Gpnmb, Ccl3 and Cathepsin D (CtsD) gene expression, and sGpnmb levels of RAW264.7 cells exposed to 10 μM U18666A and/or 250 μM NB-DNJ for 20 h. Data ( n = 3 mean ± S.E.M.) were analysed using an unpaired t-test. * P

    Techniques Used: Inhibition, Expressing

    Gpnmb gene and protein expression are increased in macrophages exposed to U18666A. RAW264.7 murine macrophages were exposed to 1, 5 and 10 μM of U18666A for 24 h. (A) Gpnmb mRNA levels and (B) Gpnmb protein levels. (C) Soluble Gpnmb assayed by ELISA of the cell culture medium. Data ( n = 3 mean ± S.E.M.) were analysed using an unpaired t-test. * P
    Figure Legend Snippet: Gpnmb gene and protein expression are increased in macrophages exposed to U18666A. RAW264.7 murine macrophages were exposed to 1, 5 and 10 μM of U18666A for 24 h. (A) Gpnmb mRNA levels and (B) Gpnmb protein levels. (C) Soluble Gpnmb assayed by ELISA of the cell culture medium. Data ( n = 3 mean ± S.E.M.) were analysed using an unpaired t-test. * P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

    Correlation between glycosphingolipids and sGpnmb levels. Correlation between cell lysate (A) glucosylceramide (GlcCer) or (B) lactosylceramide (LacCer) and sGpnmb from the medium of RAW264.7 cells exposed to 1.25, 2.5, 5 and 10 μM of U18666A for 20 h. Correlation between tissue lysate (C) hepatic or (D) splenic GlcCer levels and plasma sGpnmb of Npc1 nih/nih mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Correlation between plasma (E) GlcCer or (F) chitotriosidase activity and sGpnmb levels from diagnosed NPC patients ( n = 17). Pearson correlational analysis was used to evaluate correlation.
    Figure Legend Snippet: Correlation between glycosphingolipids and sGpnmb levels. Correlation between cell lysate (A) glucosylceramide (GlcCer) or (B) lactosylceramide (LacCer) and sGpnmb from the medium of RAW264.7 cells exposed to 1.25, 2.5, 5 and 10 μM of U18666A for 20 h. Correlation between tissue lysate (C) hepatic or (D) splenic GlcCer levels and plasma sGpnmb of Npc1 nih/nih mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Correlation between plasma (E) GlcCer or (F) chitotriosidase activity and sGpnmb levels from diagnosed NPC patients ( n = 17). Pearson correlational analysis was used to evaluate correlation.

    Techniques Used: Mouse Assay, Activity Assay

    3) Product Images from "The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux"

    Article Title: The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2018.03.001

    Increased GPMV formation preceeds efflux of cholesterol in an NPC1 model. A: GM03123 were loaded with TopFluor-cholesterol and treated as indicated. Measurement of fluorescence in media shows an increase in cholesterol efflux elicited by MβCD treatment and inhibition by U18666A. B: Quantification of intracellular cholesterol in GM03123 fibroblasts treated with MβCD for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. C: Quantification of intracellular cholesterol in GM03123 treated with LXR agonist GW3965 for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. D: Cells treated with GW3965 for 24 hours show an increase in GPMV formation. Cells stained with filipin. Arrows indicate GPMVs. All error bars indicate the standard error of the mean.
    Figure Legend Snippet: Increased GPMV formation preceeds efflux of cholesterol in an NPC1 model. A: GM03123 were loaded with TopFluor-cholesterol and treated as indicated. Measurement of fluorescence in media shows an increase in cholesterol efflux elicited by MβCD treatment and inhibition by U18666A. B: Quantification of intracellular cholesterol in GM03123 fibroblasts treated with MβCD for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. C: Quantification of intracellular cholesterol in GM03123 treated with LXR agonist GW3965 for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. D: Cells treated with GW3965 for 24 hours show an increase in GPMV formation. Cells stained with filipin. Arrows indicate GPMVs. All error bars indicate the standard error of the mean.

    Techniques Used: Fluorescence, Inhibition, Staining

    GPMV formation as a visible readout of cholesterol efflux. A: Treatment of cells with U18666A to aggregate cholesterol intracellularly inhibits GPMV formation. Treatment with water-soluble cholesterol (cholesterol-MβCD) to augment the cholesterol pool increases GPMV formation. Cells labeled with filipin. GPMVs indicated by arrows. B: ) and the corrected total cell fluorescence for cells treated with ApoA1 at 48 hours is shown. Error bars indicate the standard error of the mean. C. HeLa treated with ApoA1 for 24 and 48 hours show increased GPMV formation (arrows) most prominently at 24 hours, and decreased cellular cholesterol levels at 48 hours. Cells labeled with filipin.
    Figure Legend Snippet: GPMV formation as a visible readout of cholesterol efflux. A: Treatment of cells with U18666A to aggregate cholesterol intracellularly inhibits GPMV formation. Treatment with water-soluble cholesterol (cholesterol-MβCD) to augment the cholesterol pool increases GPMV formation. Cells labeled with filipin. GPMVs indicated by arrows. B: ) and the corrected total cell fluorescence for cells treated with ApoA1 at 48 hours is shown. Error bars indicate the standard error of the mean. C. HeLa treated with ApoA1 for 24 and 48 hours show increased GPMV formation (arrows) most prominently at 24 hours, and decreased cellular cholesterol levels at 48 hours. Cells labeled with filipin.

    Techniques Used: Labeling, Fluorescence

    4) Product Images from "Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation"

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709057

    Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.
    Figure Legend Snippet: Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.

    Techniques Used: Activation Assay, Incubation, Infection, Transfection

    Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P
    Figure Legend Snippet: Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P

    Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P
    Figure Legend Snippet: Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P

    Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Microscopy

    ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P
    Figure Legend Snippet: ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P

    Techniques Used: Labeling, Staining, Transfection

    5) Product Images from "Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators"

    Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004390

    Lipid transport out of MVBs is dispensable for CCHFV entry. (A) SW13 cells were pretreated with U18666A (30 µM) for 1 h or left untreated (mock). Then, the cells were incubated with CCHFV in the presence of the drug for 24 h and subsequently fixed, permeabilized, and stained with anti-N antibody (red), anti-CD63 antibody (green), and CellMask blue dye (grey) to define cell boundaries. The samples were imaged by immunofluorescence, and an optical section through the middle of the cell is shown (left and middle panels). Relative infection efficiencies were calculated by dividing the number of infected cells by the total number of cells and are averages of three independent experiments, with error bars representing standard deviations (right panel). (B) Cells treated as described in (A) were fixed 1 h after treatment and then stained with anti-CD63 antibody (green), filipin III (red), and CellMask red dye (grey). The images were generated as described above. (C) SW13 cells were treated with U18666A (30 µM) for 1 h or left untreated (mock), then incubated with VSV-CCHFVG, VSV-EBOVGP, or VSV-LASVGP. Luciferase activity was measured 24 h after pseudotype addition.
    Figure Legend Snippet: Lipid transport out of MVBs is dispensable for CCHFV entry. (A) SW13 cells were pretreated with U18666A (30 µM) for 1 h or left untreated (mock). Then, the cells were incubated with CCHFV in the presence of the drug for 24 h and subsequently fixed, permeabilized, and stained with anti-N antibody (red), anti-CD63 antibody (green), and CellMask blue dye (grey) to define cell boundaries. The samples were imaged by immunofluorescence, and an optical section through the middle of the cell is shown (left and middle panels). Relative infection efficiencies were calculated by dividing the number of infected cells by the total number of cells and are averages of three independent experiments, with error bars representing standard deviations (right panel). (B) Cells treated as described in (A) were fixed 1 h after treatment and then stained with anti-CD63 antibody (green), filipin III (red), and CellMask red dye (grey). The images were generated as described above. (C) SW13 cells were treated with U18666A (30 µM) for 1 h or left untreated (mock), then incubated with VSV-CCHFVG, VSV-EBOVGP, or VSV-LASVGP. Luciferase activity was measured 24 h after pseudotype addition.

    Techniques Used: Incubation, Staining, Immunofluorescence, Infection, Generated, Luciferase, Activity Assay

    6) Product Images from "Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection"

    Article Title: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection

    Journal: eLife

    doi: 10.7554/eLife.12177

    Chemical structures of U18666A and derivatives used in this study. Inhibitory constant (K i ) values denote the concentration at which each compound inhibited the incorporation of [ 14 C]oleate into cholesteryl [ 14 C]oleate by 50% in monolayers of intact Chinese Hamster Ovary 7 (CHO-7) cells that were incubated with 10% fetal calf serum (FCS) (mean of 3–14 experiments for each compound). Assays were carried out under conditions identical to those described in Figure 3A . DOI: http://dx.doi.org/10.7554/eLife.12177.004
    Figure Legend Snippet: Chemical structures of U18666A and derivatives used in this study. Inhibitory constant (K i ) values denote the concentration at which each compound inhibited the incorporation of [ 14 C]oleate into cholesteryl [ 14 C]oleate by 50% in monolayers of intact Chinese Hamster Ovary 7 (CHO-7) cells that were incubated with 10% fetal calf serum (FCS) (mean of 3–14 experiments for each compound). Assays were carried out under conditions identical to those described in Figure 3A . DOI: http://dx.doi.org/10.7554/eLife.12177.004

    Techniques Used: Concentration Assay, Incubation

    Ultraviolet (UV) crosslinking of U-X to transfected wild-type Niemann-Pick C1 (NPC1), mutant versions of NPC1, and wild-type NPC1L1 in 10–3 cells that lack NPC1. ( A and B ) Crosslinking, fluorescent labeling, and in-gel fluorescence. On day 0, 10–3 cells were set up in a six-well plate with 2 ml medium A with 5% lipoprotein-deficient serum (LPDS) per 35-mm well as described in Materials and methods. On day 1, cells were transfected by direct addition of 1 µg DNA per dish (FuGENE HD reagent) with one of the following plasmids: pcDNA3.1 control plasmid (lanes 1, 2, 9, 10); pCMV-NPC1-Flag-TEV-StrepTactin (lanes 3, 4, 11, 12); pCMV-NPC1(P691S)-Flag-TEV-StrepTactin (lanes 5, 6); pCMV-NPC1(P202A/F203A)-Flag-TEV-StrepTactin (lanes 7, 8); pCMV-NPC1L1-Flag-TEV-StrepTactin (lanes 13, 14). On day 3, all cells were incubated (without change of media) for 1 hr with 0.3 µM U-X crosslinker in the absence (–) or presence (+) of 6 µM U18666A, after which they were exposed to UV light. Cell extracts were prepared, and the crosslinked U-X was fluorescently tagged using click chemistry, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel fluorescence as in Figure 5 . ( C ) Cholesterol esterification. On day 0, 10–3 cells were set up in medium A with 5% fetal calf serum (FCS) at 2.5 x 10 5 cells/60-mm dish. On day 1, monolayers were washed once with Dulbecco’s phosphate-buffered saline (PBS), switched to fresh medium A with 5% LPDS (devoid of penicillin and streptomycin sulfate), and then transfected with 2 µg DNA per dish with the indicated plasmids as described above. After incubation for 24 hr, cells were washed once with PBS and switched to medium A with 5% LPDS containing 10 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 10% LPDS or 10% FCS as indicated. After incubation for 3 hr at 37°C, each cell monolayer was pulse-labeled for 1 hr with 0.1 mM sodium [ 14 C]oleate (5436 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the mean of triplicate incubations with individual values shown. The cellular content of [ 14 C]triglycerides in all transfected cell lines did not differ significantly in cells treated with LPDS (81–91 nmol/hr/mg) or FCS (88–105 nmol/hr/mg). Inset shows immunoblot analysis of whole cell extracts (6 µg) from the indicated transfection using a 1:1000 dilution of anti-Flag and anti-β-actin. DOI: http://dx.doi.org/10.7554/eLife.12177.009
    Figure Legend Snippet: Ultraviolet (UV) crosslinking of U-X to transfected wild-type Niemann-Pick C1 (NPC1), mutant versions of NPC1, and wild-type NPC1L1 in 10–3 cells that lack NPC1. ( A and B ) Crosslinking, fluorescent labeling, and in-gel fluorescence. On day 0, 10–3 cells were set up in a six-well plate with 2 ml medium A with 5% lipoprotein-deficient serum (LPDS) per 35-mm well as described in Materials and methods. On day 1, cells were transfected by direct addition of 1 µg DNA per dish (FuGENE HD reagent) with one of the following plasmids: pcDNA3.1 control plasmid (lanes 1, 2, 9, 10); pCMV-NPC1-Flag-TEV-StrepTactin (lanes 3, 4, 11, 12); pCMV-NPC1(P691S)-Flag-TEV-StrepTactin (lanes 5, 6); pCMV-NPC1(P202A/F203A)-Flag-TEV-StrepTactin (lanes 7, 8); pCMV-NPC1L1-Flag-TEV-StrepTactin (lanes 13, 14). On day 3, all cells were incubated (without change of media) for 1 hr with 0.3 µM U-X crosslinker in the absence (–) or presence (+) of 6 µM U18666A, after which they were exposed to UV light. Cell extracts were prepared, and the crosslinked U-X was fluorescently tagged using click chemistry, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel fluorescence as in Figure 5 . ( C ) Cholesterol esterification. On day 0, 10–3 cells were set up in medium A with 5% fetal calf serum (FCS) at 2.5 x 10 5 cells/60-mm dish. On day 1, monolayers were washed once with Dulbecco’s phosphate-buffered saline (PBS), switched to fresh medium A with 5% LPDS (devoid of penicillin and streptomycin sulfate), and then transfected with 2 µg DNA per dish with the indicated plasmids as described above. After incubation for 24 hr, cells were washed once with PBS and switched to medium A with 5% LPDS containing 10 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 10% LPDS or 10% FCS as indicated. After incubation for 3 hr at 37°C, each cell monolayer was pulse-labeled for 1 hr with 0.1 mM sodium [ 14 C]oleate (5436 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the mean of triplicate incubations with individual values shown. The cellular content of [ 14 C]triglycerides in all transfected cell lines did not differ significantly in cells treated with LPDS (81–91 nmol/hr/mg) or FCS (88–105 nmol/hr/mg). Inset shows immunoblot analysis of whole cell extracts (6 µg) from the indicated transfection using a 1:1000 dilution of anti-Flag and anti-β-actin. DOI: http://dx.doi.org/10.7554/eLife.12177.009

    Techniques Used: Transfection, Mutagenesis, Labeling, Fluorescence, Plasmid Preparation, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

    Ultraviolet (UV) crosslinking of U-X to Niemann-Pick C1 (NPC1) in Chinese Hamster Ovary (CHO)-K1 cells, but not in mutant 10–3 cells. On day 0, CHO-K1 cells and 10–3 cells (mutant derivative of CHO-K1 cells that lack NPC1) were set up in a six-well plate in medium A with 5% lipoprotein-deficient serum (LPDS) (2 ml/35-mm well). ( A ) In-gel fluorescence of U-X binding proteins in parental CHO-K1 cells and mutant 10–3 cells that lack NPC1. On day 3, each well of cells received a direct addition of ethanol (final concentration, 0.2%) containing 0.3 µM of U-X crosslinker (lanes 1–9) and one of the following compounds: none (lanes 1, 4, 7); 6 µM U18666A (lanes 2, 5, 8); or 6 µM compound A (lanes 3, 6, 9). After incubation for 1 hr at 37°C, cells in lanes 4–9 were exposed to UV light as described in Materials and methods. Cell extracts were prepared, and the crosslinked U-X was fluorescently tagged using click chemistry. Proteins were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by in-gel fluorescence scanning. Arrow denotes a 190-kDa protein crosslinked to U-X and competed by U18666A, but not compound A . ( B ) Fluorescent labeling of 190-kDa protein in wild-type (WT) CHO-K1 cells, but not in 10–3 cells lacking NPC1: restoration by expression of NPC1. On day 1, mutant 10–3 cells were transfected with 1 µg/well of either control plasmid (pcDNA3.1; lane 12) or plasmid encoding NPC1 (pCMV-NPC1-Flag-TEV-StrepTactin; lane 13). Nontransfected CHO-K1 and 10–3 cells were set up in parallel (lanes 10 and 11, respectively). On day 3, all cells were incubated with 0.3 µM U-X for 1 hr, after which they were exposed to UV light. Cell extracts were prepared, and the cross-linked U-X was fluorescently tagged using click chemistry, followed by SDS-PAGE and in-gel fluorescence as in Panel A. Closed arrow denotes a 190-kDa protein crosslinked to U-X ; open arrow shows the appearance of a similar band in transfected 10–3 cells expressing epitope-tagged NPC1. DOI: http://dx.doi.org/10.7554/eLife.12177.007
    Figure Legend Snippet: Ultraviolet (UV) crosslinking of U-X to Niemann-Pick C1 (NPC1) in Chinese Hamster Ovary (CHO)-K1 cells, but not in mutant 10–3 cells. On day 0, CHO-K1 cells and 10–3 cells (mutant derivative of CHO-K1 cells that lack NPC1) were set up in a six-well plate in medium A with 5% lipoprotein-deficient serum (LPDS) (2 ml/35-mm well). ( A ) In-gel fluorescence of U-X binding proteins in parental CHO-K1 cells and mutant 10–3 cells that lack NPC1. On day 3, each well of cells received a direct addition of ethanol (final concentration, 0.2%) containing 0.3 µM of U-X crosslinker (lanes 1–9) and one of the following compounds: none (lanes 1, 4, 7); 6 µM U18666A (lanes 2, 5, 8); or 6 µM compound A (lanes 3, 6, 9). After incubation for 1 hr at 37°C, cells in lanes 4–9 were exposed to UV light as described in Materials and methods. Cell extracts were prepared, and the crosslinked U-X was fluorescently tagged using click chemistry. Proteins were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by in-gel fluorescence scanning. Arrow denotes a 190-kDa protein crosslinked to U-X and competed by U18666A, but not compound A . ( B ) Fluorescent labeling of 190-kDa protein in wild-type (WT) CHO-K1 cells, but not in 10–3 cells lacking NPC1: restoration by expression of NPC1. On day 1, mutant 10–3 cells were transfected with 1 µg/well of either control plasmid (pcDNA3.1; lane 12) or plasmid encoding NPC1 (pCMV-NPC1-Flag-TEV-StrepTactin; lane 13). Nontransfected CHO-K1 and 10–3 cells were set up in parallel (lanes 10 and 11, respectively). On day 3, all cells were incubated with 0.3 µM U-X for 1 hr, after which they were exposed to UV light. Cell extracts were prepared, and the cross-linked U-X was fluorescently tagged using click chemistry, followed by SDS-PAGE and in-gel fluorescence as in Panel A. Closed arrow denotes a 190-kDa protein crosslinked to U-X ; open arrow shows the appearance of a similar band in transfected 10–3 cells expressing epitope-tagged NPC1. DOI: http://dx.doi.org/10.7554/eLife.12177.007

    Techniques Used: Mutagenesis, Fluorescence, Binding Assay, Concentration Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Labeling, Expressing, Transfection, Plasmid Preparation

    Cholesterol esterification, sterol regulatory element-binding protein (SREBP) 2 processing, and 125 I-LDL (low density lipoprotein) degradation in Chinese Hamster Ovary (CHO) 7 cells. On day 0, CHO-7 cells were set up for experiments in medium A with 5% lipoprotein-deficient serum (LPDS) at a density of 2.5 x 10 5 cells/60-mm dish. On day 2, the medium was switched to fresh medium containing 5 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the medium was switched to medium B containing either 10% LPDS or 10% fetal calf serum (FCS), 50 µM compactin, 50 µM mevalonate, and various concentrations of the indicated compound and then incubated for either 7 hr ( A ) or 6 hr ( B ) as described below. ( A ) Cholesterol esterification. After a 5 hr incubation with the above medium with 10% FCS and the indicated compounds, each cell monolayer was labeled for 2 hr with 0.2 mM sodium [ 14 C]oleate (8133 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the average of duplicate incubations. Values for [ 14 C]triglyceride content in cells treated with 1 µM of U18666A, compound A , and U-X were 117, 116, and 106 nmol/hr/ mg protein, respectively. (B) SREBP-2 processing. After a 5 hr incubation with the above medium containing either 10% LPDS (lane 1) or 10% FCS (lanes 2–11), each monolayer received a direct addition of 20 µg/ml of N-acetyl-leu-leu-norleucinal (A.G. Scientific, San Diego, CA). After 1 hr, cells were harvested and fractionated into a nuclear extract and 10 5 g membrane fraction ( Sakai et al., 1996 ). Aliquots (30 and 10 μg protein for SREBP-2 and Niemann-Pick C1 [NPC1], respectively) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblot analysis and image scanning were carried out with monoclonal antibodies directed against SREBP-2 or NPC1 as described in Materials and methods. ( C ) 125 I-LDL degradation. On day 3, the medium was switched to medium B containing 5% human LPDS, 20 µg protein/ml of either 125 I-LDL (for 125 I-LDL degradation) or unlabeled LDL (for cholesterol esterification), 10 µM compactin, and 50 µM mevalonate in the presence of one of the following compounds: none, 0.3 µM U18666A, 0.3 µM U-X , and 50 µM chloroquine. For the 125 I-LDL degradation assay, cells were incubated for 6 hr with 125 I-LDL (48 cpm/ng protein), after which the medium from each monolayer was removed and its content of 125 I-monoiodotyrosine was measured as previously described ( Goldstein, 1983 ). The 100% control value for 125 I-LDL degradation in the absence of any compound (none) was 4.1 µg/6 hr/mg of protein. The cholesterol esterification assay was carried out as in A except that the cells were pulse-labeled with 0.1 mM [ 14 C]oleate (6515 dpm/nmol). The 100% control value for cholesteryl [ 14 C]oleate formed was 5.9 nmol/hr/mg protein. The content of [ 14 C]triglycerides in cells receiving the various compounds were not significantly different: 50, 57, 55, and 81 nmol/hr/mg protein, respectively, for no addition, U18666A, U-X , and chloroquine. All values are the mean of triplicate incubations with individual values shown. DOI: http://dx.doi.org/10.7554/eLife.12177.005
    Figure Legend Snippet: Cholesterol esterification, sterol regulatory element-binding protein (SREBP) 2 processing, and 125 I-LDL (low density lipoprotein) degradation in Chinese Hamster Ovary (CHO) 7 cells. On day 0, CHO-7 cells were set up for experiments in medium A with 5% lipoprotein-deficient serum (LPDS) at a density of 2.5 x 10 5 cells/60-mm dish. On day 2, the medium was switched to fresh medium containing 5 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the medium was switched to medium B containing either 10% LPDS or 10% fetal calf serum (FCS), 50 µM compactin, 50 µM mevalonate, and various concentrations of the indicated compound and then incubated for either 7 hr ( A ) or 6 hr ( B ) as described below. ( A ) Cholesterol esterification. After a 5 hr incubation with the above medium with 10% FCS and the indicated compounds, each cell monolayer was labeled for 2 hr with 0.2 mM sodium [ 14 C]oleate (8133 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the average of duplicate incubations. Values for [ 14 C]triglyceride content in cells treated with 1 µM of U18666A, compound A , and U-X were 117, 116, and 106 nmol/hr/ mg protein, respectively. (B) SREBP-2 processing. After a 5 hr incubation with the above medium containing either 10% LPDS (lane 1) or 10% FCS (lanes 2–11), each monolayer received a direct addition of 20 µg/ml of N-acetyl-leu-leu-norleucinal (A.G. Scientific, San Diego, CA). After 1 hr, cells were harvested and fractionated into a nuclear extract and 10 5 g membrane fraction ( Sakai et al., 1996 ). Aliquots (30 and 10 μg protein for SREBP-2 and Niemann-Pick C1 [NPC1], respectively) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblot analysis and image scanning were carried out with monoclonal antibodies directed against SREBP-2 or NPC1 as described in Materials and methods. ( C ) 125 I-LDL degradation. On day 3, the medium was switched to medium B containing 5% human LPDS, 20 µg protein/ml of either 125 I-LDL (for 125 I-LDL degradation) or unlabeled LDL (for cholesterol esterification), 10 µM compactin, and 50 µM mevalonate in the presence of one of the following compounds: none, 0.3 µM U18666A, 0.3 µM U-X , and 50 µM chloroquine. For the 125 I-LDL degradation assay, cells were incubated for 6 hr with 125 I-LDL (48 cpm/ng protein), after which the medium from each monolayer was removed and its content of 125 I-monoiodotyrosine was measured as previously described ( Goldstein, 1983 ). The 100% control value for 125 I-LDL degradation in the absence of any compound (none) was 4.1 µg/6 hr/mg of protein. The cholesterol esterification assay was carried out as in A except that the cells were pulse-labeled with 0.1 mM [ 14 C]oleate (6515 dpm/nmol). The 100% control value for cholesteryl [ 14 C]oleate formed was 5.9 nmol/hr/mg protein. The content of [ 14 C]triglycerides in cells receiving the various compounds were not significantly different: 50, 57, 55, and 81 nmol/hr/mg protein, respectively, for no addition, U18666A, U-X , and chloroquine. All values are the mean of triplicate incubations with individual values shown. DOI: http://dx.doi.org/10.7554/eLife.12177.005

    Techniques Used: Binding Assay, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, SDS Page, Degradation Assay

    Cholesterol esterification in Chinese Hamster Ovary (CHO) 7 and TR-4139 cells that overexpress Niemann-Pick C1 (NPC1). CHO-7 cells and TR-4139 cells were set up for experiments in medium A with 5% lipoprotein-deficient serum (LPDS) as described in the legend to Figure 3 . On day 2, the medium was switched to fresh medium containing 5 µM sodium compactin, 50 µM sodium mevalonate, and the indicated concentration of U18666A. After incubation for 4 hr, each cell monolayer was labeled for 2 hr with 0.2 mM sodium [ 14 C]oleate (11,662 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the average of duplicate incubations. The cellular content of [ 14 C]triglycerides in CHO-7 and TR-4139 cells was 38 and 40 nmol/hr/mg protein, respectively. Inset shows immunoblots of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels of whole cell extracts (30 µg) from the indicated cell line incubated with 0.5 µg/ml anti-NPC1 or 1 µg/ml anti-calnexin as described in Materials and methods. DOI: http://dx.doi.org/10.7554/eLife.12177.006
    Figure Legend Snippet: Cholesterol esterification in Chinese Hamster Ovary (CHO) 7 and TR-4139 cells that overexpress Niemann-Pick C1 (NPC1). CHO-7 cells and TR-4139 cells were set up for experiments in medium A with 5% lipoprotein-deficient serum (LPDS) as described in the legend to Figure 3 . On day 2, the medium was switched to fresh medium containing 5 µM sodium compactin, 50 µM sodium mevalonate, and the indicated concentration of U18666A. After incubation for 4 hr, each cell monolayer was labeled for 2 hr with 0.2 mM sodium [ 14 C]oleate (11,662 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the average of duplicate incubations. The cellular content of [ 14 C]triglycerides in CHO-7 and TR-4139 cells was 38 and 40 nmol/hr/mg protein, respectively. Inset shows immunoblots of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels of whole cell extracts (30 µg) from the indicated cell line incubated with 0.5 µg/ml anti-NPC1 or 1 µg/ml anti-calnexin as described in Materials and methods. DOI: http://dx.doi.org/10.7554/eLife.12177.006

    Techniques Used: Concentration Assay, Incubation, Labeling, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page

    7) Product Images from "Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis"

    Article Title: Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

    Journal: Orphanet Journal of Rare Diseases

    doi: 10.1186/s13023-017-0697-y

    Induction of gliosis by U18666A. a Unaffected control cells were treated with ( b ) U18666A to induce cholesterol accumulations shown by Filipin staining (blue). Scale 100 μm. c Calculation of relative fluorescence confirmed higher cholesterol amount in U18666A treated cells ( N = 4, n = 6–8). d Increased cholesterol amount in U18666A treated cells was also detected by means of the Amplex red assay ( N = 4, n = 7–8). e FACS analysis of GFAP + /vimentin + cells and f GFAP + /Ki67 + cells revealed an increased amount of reactive astrocytes ( N = 4, n = 7–12). g Treatment with U18666A resulted in a reduced amount of p-vimentin and ( h ) p-GFAP ( N = 4, n = 8–12)
    Figure Legend Snippet: Induction of gliosis by U18666A. a Unaffected control cells were treated with ( b ) U18666A to induce cholesterol accumulations shown by Filipin staining (blue). Scale 100 μm. c Calculation of relative fluorescence confirmed higher cholesterol amount in U18666A treated cells ( N = 4, n = 6–8). d Increased cholesterol amount in U18666A treated cells was also detected by means of the Amplex red assay ( N = 4, n = 7–8). e FACS analysis of GFAP + /vimentin + cells and f GFAP + /Ki67 + cells revealed an increased amount of reactive astrocytes ( N = 4, n = 7–12). g Treatment with U18666A resulted in a reduced amount of p-vimentin and ( h ) p-GFAP ( N = 4, n = 8–12)

    Techniques Used: Staining, Fluorescence, Amplex Red Assay, FACS

    8) Product Images from "ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease"

    Article Title: ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005193

    Constitutively active ARF6 increases cholesterol removal in NPC-like cells. A , HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B , HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C , Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods ). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).
    Figure Legend Snippet: Constitutively active ARF6 increases cholesterol removal in NPC-like cells. A , HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B , HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C , Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods ). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).

    Techniques Used: Staining, Transfection, Mutagenesis, Expressing, Quantitation Assay, Two Tailed Test

    9) Product Images from "Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic"

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

    Journal: mBio

    doi: 10.1128/mBio.02313-16

    Altered cellular cholesterol homeostasis is bactericidal. (A) Microscopy images showing that U18666A treatment traps cholesterol in the C. burnetii PV in HeLa cells. mCherry- C. burnetii -infected HeLa cells were treated with 5 µM U18666A for 6 h, fixed, and stained for sterols (filipin) and PV (LAMP-1). Compared to mock-treated cells, there is an increase in filipin labeling in and around the PV following treatment with U18666A. The white arrows point to the PVs. Filipin intensity is shown as a heat map, with yellow showing the highest filipin intensity and blue showing the lowest filipin intensity. Bars = 5 µm. (B) Quantitation of lytic PVs in U18666A-treated cells after treatment of mCherry- C. burnetii- infected HeLa cells with 1 or 5 µM U18666A. PVs were scored for the presence (lytic) or absence (nonlytic) of free mCherry in the PV lumen, resulting from the lysis of mCherry-expressing bacteria. The means plus SEM (error bars) from three individual experiments are shown. The means were compared to the value with no cholesterol by one-way ANOVA with Dunnett’s posthoc test, and statistically different values are indicated by asterisks as follows: *, P
    Figure Legend Snippet: Altered cellular cholesterol homeostasis is bactericidal. (A) Microscopy images showing that U18666A treatment traps cholesterol in the C. burnetii PV in HeLa cells. mCherry- C. burnetii -infected HeLa cells were treated with 5 µM U18666A for 6 h, fixed, and stained for sterols (filipin) and PV (LAMP-1). Compared to mock-treated cells, there is an increase in filipin labeling in and around the PV following treatment with U18666A. The white arrows point to the PVs. Filipin intensity is shown as a heat map, with yellow showing the highest filipin intensity and blue showing the lowest filipin intensity. Bars = 5 µm. (B) Quantitation of lytic PVs in U18666A-treated cells after treatment of mCherry- C. burnetii- infected HeLa cells with 1 or 5 µM U18666A. PVs were scored for the presence (lytic) or absence (nonlytic) of free mCherry in the PV lumen, resulting from the lysis of mCherry-expressing bacteria. The means plus SEM (error bars) from three individual experiments are shown. The means were compared to the value with no cholesterol by one-way ANOVA with Dunnett’s posthoc test, and statistically different values are indicated by asterisks as follows: *, P

    Techniques Used: Microscopy, Infection, Staining, Labeling, Quantitation Assay, Lysis, Expressing

    10) Product Images from "Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies"

    Article Title: Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies

    Journal: Journal of Virology

    doi: 10.1128/JVI.03109-15

    Inhibition of MVB morphogenesis impairs the release of filaments but not the release of spheres. (A) p1.2×HBVΔCore-transfected Huh 7.5 cells were treated with different concentrations of MVBs inhibitor U18666A (U18). The lysates (LS) and
    Figure Legend Snippet: Inhibition of MVB morphogenesis impairs the release of filaments but not the release of spheres. (A) p1.2×HBVΔCore-transfected Huh 7.5 cells were treated with different concentrations of MVBs inhibitor U18666A (U18). The lysates (LS) and

    Techniques Used: Inhibition, Transfection

    11) Product Images from "Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content"

    Article Title: Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050262

    Manipulation of lysosomal cholesterol content modulates the cellular sensitivity to apoptosis. Cholesterol content of human fibroblasts was modulated using U18666A, quinacrine, methyl-β-cyclodextrin (MβCD) or 25-hydroxy cholesterol (25-HC) before apoptosis was induced using O-methyl-serine dodecylamide hydrochloride (MSDH; 24 h). Phase contrast images of A ) wt and B ) NPC1-mutant fibroblasts (NPC1 mut ). Scale bar 20 µm. C ) Viability of cultures in A and B, respectively, assessed by the MTT assay (n = 4). Viability is expressed as percentage of untreated cultures. D ) Caspase-3 like activity (n = 4–8). E ) Representative curve of increase in green fluorescence during photo-oxidation of acridine orange. F ) Quantification of lag time (as presented in E; n = 5–6). Data are presented as the mean ± SD, * p≤0.05.
    Figure Legend Snippet: Manipulation of lysosomal cholesterol content modulates the cellular sensitivity to apoptosis. Cholesterol content of human fibroblasts was modulated using U18666A, quinacrine, methyl-β-cyclodextrin (MβCD) or 25-hydroxy cholesterol (25-HC) before apoptosis was induced using O-methyl-serine dodecylamide hydrochloride (MSDH; 24 h). Phase contrast images of A ) wt and B ) NPC1-mutant fibroblasts (NPC1 mut ). Scale bar 20 µm. C ) Viability of cultures in A and B, respectively, assessed by the MTT assay (n = 4). Viability is expressed as percentage of untreated cultures. D ) Caspase-3 like activity (n = 4–8). E ) Representative curve of increase in green fluorescence during photo-oxidation of acridine orange. F ) Quantification of lag time (as presented in E; n = 5–6). Data are presented as the mean ± SD, * p≤0.05.

    Techniques Used: Mutagenesis, MTT Assay, Activity Assay, Fluorescence

    Cholesterol Accumulation in Cortical Neurons Rescues Cells from Apoptosis Induced by MSDH and Oxidative Stress. Cortical neurons were treated with U18666A. A ) Filipin staining and differential interference contrast microscopy (DIC) images (scale bar 10 µm) and B ) a higher magnification of filipin staining (scale bar 10 μM). C ) Viability analysis and caspase-3-like activity (n = 3) after 72 h. D ) Phase contrast images (scale bar 20 µm) and E ) viability analysis (MTT assay; n = 3) of cultures exposed to O-methyl-serine dodecylamide hydrochloride (MSDH) or H 2 O 2 , generated by glucose oxidase, with or without pretreatment with U18666A (48 h). Viability is expressed as percentage of untreated control. Data are presented as the mean ± SD, * p≤0.05, ns; non-significant.
    Figure Legend Snippet: Cholesterol Accumulation in Cortical Neurons Rescues Cells from Apoptosis Induced by MSDH and Oxidative Stress. Cortical neurons were treated with U18666A. A ) Filipin staining and differential interference contrast microscopy (DIC) images (scale bar 10 µm) and B ) a higher magnification of filipin staining (scale bar 10 μM). C ) Viability analysis and caspase-3-like activity (n = 3) after 72 h. D ) Phase contrast images (scale bar 20 µm) and E ) viability analysis (MTT assay; n = 3) of cultures exposed to O-methyl-serine dodecylamide hydrochloride (MSDH) or H 2 O 2 , generated by glucose oxidase, with or without pretreatment with U18666A (48 h). Viability is expressed as percentage of untreated control. Data are presented as the mean ± SD, * p≤0.05, ns; non-significant.

    Techniques Used: Staining, Microscopy, Activity Assay, MTT Assay, Generated

    Cholesterol, and not accumulating sphingolipids, is responsible for the apoptosis protection. Human wt fibroblasts, with or without U18666A treatment, and NPC1-mutant fibroblasts were treated with vehicle (dimethyl sulfoxide; DMSO) or myriocin to inhibit sphingolipid biosynthesis. A ) Sphingomyelin (n = 3), B ) cholesterol content (n = 4) and C ) filipin staining (scale bar 10 μm) of human fibroblasts. D ) Phase contrast images of human fibroblasts exposed to O-methyl-serine dodecylamide hydrochloride (MSDH; 24 h). Scale bar 20 μm. E ) Viability of cultures in D, assessed by the MTT assay (n = 3). Viability is expressed as percentage of MSDH-untreated cultures. Data are presented as the mean ± SD, * p≤0.05, ns; non-significant.
    Figure Legend Snippet: Cholesterol, and not accumulating sphingolipids, is responsible for the apoptosis protection. Human wt fibroblasts, with or without U18666A treatment, and NPC1-mutant fibroblasts were treated with vehicle (dimethyl sulfoxide; DMSO) or myriocin to inhibit sphingolipid biosynthesis. A ) Sphingomyelin (n = 3), B ) cholesterol content (n = 4) and C ) filipin staining (scale bar 10 μm) of human fibroblasts. D ) Phase contrast images of human fibroblasts exposed to O-methyl-serine dodecylamide hydrochloride (MSDH; 24 h). Scale bar 20 μm. E ) Viability of cultures in D, assessed by the MTT assay (n = 3). Viability is expressed as percentage of MSDH-untreated cultures. Data are presented as the mean ± SD, * p≤0.05, ns; non-significant.

    Techniques Used: Mutagenesis, Staining, MTT Assay

    Cholesterol modulation in human fibroblasts is associated with alterations of the lysosomal compartment. Human wt fibroblasts were treated with U18666A or quinacrine to induce cholesterol accumulation, and NPC1-mutant fibroblasts were treated with methyl-β-cyclodextrin (MβCD) or 25-hydroxy cholesterol (25-HC) to revert cholesterol storage. A ) Measurement of unesterified cholesterol (n = 4) and B ) representative images of filipin staining (scale bar 10 µm). C and D ) Representative histogram from flow cytometric analysis of Lysotracker fluorescence staining. M1 gate denotes the highly fluorescent population. E and F ) Quantification of peak channel in the M1 population (seen in C and D; n = 4). Data are presented as the mean ± SD, * p≤0.05.
    Figure Legend Snippet: Cholesterol modulation in human fibroblasts is associated with alterations of the lysosomal compartment. Human wt fibroblasts were treated with U18666A or quinacrine to induce cholesterol accumulation, and NPC1-mutant fibroblasts were treated with methyl-β-cyclodextrin (MβCD) or 25-hydroxy cholesterol (25-HC) to revert cholesterol storage. A ) Measurement of unesterified cholesterol (n = 4) and B ) representative images of filipin staining (scale bar 10 µm). C and D ) Representative histogram from flow cytometric analysis of Lysotracker fluorescence staining. M1 gate denotes the highly fluorescent population. E and F ) Quantification of peak channel in the M1 population (seen in C and D; n = 4). Data are presented as the mean ± SD, * p≤0.05.

    Techniques Used: Mutagenesis, Staining, Flow Cytometry, Fluorescence

    Cholesterol Modulation Influences the Sensitivity of MEFs to Oxidative Stress-induced Apoptosis. Mouse embryonic fibroblasts (MEFs) deficient for both LAMP-1 and LAMP-2 (LAMP null ) were treated with U18666A or methyl-β-cyclodextrin (MβCD). A ) Filipin staining of wt and LAMP null MEFs (scale bar 10 µm). B ) Phase contrast images (scale bar 5 µm) and C ) viability (n = 4) of wt and LAMP null MEFs 24 h after H 2 O 2 exposure. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean ± SD, * p≤0.05, ns; non-significant.
    Figure Legend Snippet: Cholesterol Modulation Influences the Sensitivity of MEFs to Oxidative Stress-induced Apoptosis. Mouse embryonic fibroblasts (MEFs) deficient for both LAMP-1 and LAMP-2 (LAMP null ) were treated with U18666A or methyl-β-cyclodextrin (MβCD). A ) Filipin staining of wt and LAMP null MEFs (scale bar 10 µm). B ) Phase contrast images (scale bar 5 µm) and C ) viability (n = 4) of wt and LAMP null MEFs 24 h after H 2 O 2 exposure. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean ± SD, * p≤0.05, ns; non-significant.

    Techniques Used: Staining

    Cholesterol Accumulation Protects MEFs from Oxidative Stress-induced Apoptosis, Independent of the Expression of LAMP Proteins. A ) Localization (scale bar 10 μm) and B ) expression of lysosome-associated membrane protein-2 (LAMP-2) in wt and NPC1-mutant (NPC1 mut ) human fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to verify equal protein loading. One representative blot out of three is shown. C ) Phase contrast images (scale bar 5 µm) and D ) viability analysis (n = 4) of wt mouse embryonic fibroblasts (MEFs) and MEFs deficient for LAMP-1 (LAMP-1 −/− ) or LAMP-2 (LAMP-2 −/− ) 24 h after H 2 O 2 exposure, with or without U18666A pretreatment. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean ± SD, * p≤0.05) Filipin staining in MEFs, with or without U18666A treatment. Scale bar 10 µm.
    Figure Legend Snippet: Cholesterol Accumulation Protects MEFs from Oxidative Stress-induced Apoptosis, Independent of the Expression of LAMP Proteins. A ) Localization (scale bar 10 μm) and B ) expression of lysosome-associated membrane protein-2 (LAMP-2) in wt and NPC1-mutant (NPC1 mut ) human fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to verify equal protein loading. One representative blot out of three is shown. C ) Phase contrast images (scale bar 5 µm) and D ) viability analysis (n = 4) of wt mouse embryonic fibroblasts (MEFs) and MEFs deficient for LAMP-1 (LAMP-1 −/− ) or LAMP-2 (LAMP-2 −/− ) 24 h after H 2 O 2 exposure, with or without U18666A pretreatment. Viability was measured by crystal violet staining and expressed as percentage of untreated cultures. Data are presented as the mean ± SD, * p≤0.05) Filipin staining in MEFs, with or without U18666A treatment. Scale bar 10 µm.

    Techniques Used: Expressing, Mutagenesis, Staining

    12) Product Images from "Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *"

    Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.740928

    Movement of free cholesterol in MA-10 cells. A , confocal microscope images of cells transfected with mCherry-D4. Images a–e , time course of one control (untreated) cell; images f–j , time course of one cell treated with Bt 2 cAMP ( dbcAMP ). Scale bar, 10 μm. B , time course of fluorescence intensity at the plasma membrane of control and Bt 2 cAMP-treated cells transfected with mCherry-D4. C , time course of progesterone levels in untransfected cell and cells transfected with mCherry-D4 after treatment with Bt 2 cAMP. D , confocal microscope images of cells transfected with mCherry-D4 before treatment and 30 min after treatment with methyl-β-cyclodextrin. Scale bar, 10 μm. E , confocal microscope images of cells before treatment and cells incubated with U18666A for 4 h and then treated with Bt 2 cAMP for 2 h. Scale bar, 10 μm. F, progesterone production in mCherry-D4-transfected cells treated with U18666A, U18666A + Bt 2 cAMP, Bt 2 cAMP and control (untreated). Data represent means ± S.D. of at least three independent experiments performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (***) or t test (###) were used to calculate statistical significance; ***, ###, p
    Figure Legend Snippet: Movement of free cholesterol in MA-10 cells. A , confocal microscope images of cells transfected with mCherry-D4. Images a–e , time course of one control (untreated) cell; images f–j , time course of one cell treated with Bt 2 cAMP ( dbcAMP ). Scale bar, 10 μm. B , time course of fluorescence intensity at the plasma membrane of control and Bt 2 cAMP-treated cells transfected with mCherry-D4. C , time course of progesterone levels in untransfected cell and cells transfected with mCherry-D4 after treatment with Bt 2 cAMP. D , confocal microscope images of cells transfected with mCherry-D4 before treatment and 30 min after treatment with methyl-β-cyclodextrin. Scale bar, 10 μm. E , confocal microscope images of cells before treatment and cells incubated with U18666A for 4 h and then treated with Bt 2 cAMP for 2 h. Scale bar, 10 μm. F, progesterone production in mCherry-D4-transfected cells treated with U18666A, U18666A + Bt 2 cAMP, Bt 2 cAMP and control (untreated). Data represent means ± S.D. of at least three independent experiments performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (***) or t test (###) were used to calculate statistical significance; ***, ###, p

    Techniques Used: Microscopy, Transfection, Fluorescence, Incubation

    13) Product Images from "Histone Deacetylase Inhibition Decreases Cholesterol Levels in Neuronal Cells by Modulating Key Genes in Cholesterol Synthesis, Uptake and Efflux"

    Article Title: Histone Deacetylase Inhibition Decreases Cholesterol Levels in Neuronal Cells by Modulating Key Genes in Cholesterol Synthesis, Uptake and Efflux

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053394

    TSA treatment partially reverts the effect of U18666A on the expression of genes involved in cholesterol synthesis, uptake and efflux. SH-SY5Y neuroblastoma cells were pre-treated with 3 µg/ml U18666A for 6 h and with or without 250 nM TSA for 16 h. mRNA transcript levels of HMGCR, MVK, LDLR and ABCA1 were analyzed by qPCR. Values were normalized to the internal standard β-actin and are expressed as fold change relative to untreated cells. Data represent means ± SEM from at least three individual experiments (* p
    Figure Legend Snippet: TSA treatment partially reverts the effect of U18666A on the expression of genes involved in cholesterol synthesis, uptake and efflux. SH-SY5Y neuroblastoma cells were pre-treated with 3 µg/ml U18666A for 6 h and with or without 250 nM TSA for 16 h. mRNA transcript levels of HMGCR, MVK, LDLR and ABCA1 were analyzed by qPCR. Values were normalized to the internal standard β-actin and are expressed as fold change relative to untreated cells. Data represent means ± SEM from at least three individual experiments (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    TSA treatment partially reverts the U18666A-induced phenotype by promoting cholesterol redistribution in neuroblastoma cells. A) Filipin III, lysosome-associated membrane protein 2 (LAMP-2) and acetyl-histone 4 (AcH4) immunofluorescence staining of SH-SY5Y cells pre-treated with 1 µg/ml U18666A for 24 h and with or without 250 nM TSA for 16 h. After the 24 h treatment with U18666A, the medium was removed and replaced with new medium without U18666A, and with or without TSA. Colocalization images of filipin III and LAMP-2 immunostaining are also presented. The results shown are representative of those obtained in at least three independent experiments. ( scale bar = 40 µm). Quantification of filipin III fluorescence (B) and filipin III and LAMP-2 colocalization (C) was performed, the values were normalized to total cell number assessed by AcH4 immunostaining and are expressed as fold change relative to U18666A treated cells. Data represent means ± SEM of at least three independent experiments (* p
    Figure Legend Snippet: TSA treatment partially reverts the U18666A-induced phenotype by promoting cholesterol redistribution in neuroblastoma cells. A) Filipin III, lysosome-associated membrane protein 2 (LAMP-2) and acetyl-histone 4 (AcH4) immunofluorescence staining of SH-SY5Y cells pre-treated with 1 µg/ml U18666A for 24 h and with or without 250 nM TSA for 16 h. After the 24 h treatment with U18666A, the medium was removed and replaced with new medium without U18666A, and with or without TSA. Colocalization images of filipin III and LAMP-2 immunostaining are also presented. The results shown are representative of those obtained in at least three independent experiments. ( scale bar = 40 µm). Quantification of filipin III fluorescence (B) and filipin III and LAMP-2 colocalization (C) was performed, the values were normalized to total cell number assessed by AcH4 immunostaining and are expressed as fold change relative to U18666A treated cells. Data represent means ± SEM of at least three independent experiments (* p

    Techniques Used: Immunofluorescence, Staining, Immunostaining, Fluorescence

    TSA reverts the increase in total cholesterol levels observed after U18666A treatment. A) Filipin III immunofluorescence staining of SH-SY5Y cells treated for the indicated time-points with 1 µg/ml U18666A or vehicle ( scale bar = 40 µm). B) SH-SY5Y cells were treated with 3 µg/ml U18666A for 24 h and with or without 250 nM TSA for 48 h, and total cholesterol levels were determined. Values were normalized to total protein content and expressed as ng of cholesterol per µg of total protein. Data represent means ± SEM from at least three individual experiments (** p
    Figure Legend Snippet: TSA reverts the increase in total cholesterol levels observed after U18666A treatment. A) Filipin III immunofluorescence staining of SH-SY5Y cells treated for the indicated time-points with 1 µg/ml U18666A or vehicle ( scale bar = 40 µm). B) SH-SY5Y cells were treated with 3 µg/ml U18666A for 24 h and with or without 250 nM TSA for 48 h, and total cholesterol levels were determined. Values were normalized to total protein content and expressed as ng of cholesterol per µg of total protein. Data represent means ± SEM from at least three individual experiments (** p

    Techniques Used: Immunofluorescence, Staining

    14) Product Images from "Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy"

    Article Title: Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

    Journal: Scientific Reports

    doi: 10.1038/srep18017

    Effect of U18666A and 25-HC in the content and distribution of lysosomal sphingomyelin. ( a ) ASMase +/+ and ASMase −/− PMH were treated overnight with U18666A (1 μg/ml), 25-HC (1 μg/ml) or both and lysosomal colocalization (Lamp2 staining) with cholesterol (filipin staining) and sphingomyelin (lysenin staining) was analysed by inmunofluorescence. ( b,c ) Total sphingomyelin levels in PMH following the different treatments were analysed with a colorimetric detection kit. Scale bar represents 20 μm.
    Figure Legend Snippet: Effect of U18666A and 25-HC in the content and distribution of lysosomal sphingomyelin. ( a ) ASMase +/+ and ASMase −/− PMH were treated overnight with U18666A (1 μg/ml), 25-HC (1 μg/ml) or both and lysosomal colocalization (Lamp2 staining) with cholesterol (filipin staining) and sphingomyelin (lysenin staining) was analysed by inmunofluorescence. ( b,c ) Total sphingomyelin levels in PMH following the different treatments were analysed with a colorimetric detection kit. Scale bar represents 20 μm.

    Techniques Used: Staining

    Effect of U18666A and 25-HC in lysosomal colocalization with mitochondria and APAP susceptibility ( a ) ASMase +/+ PMH were pretreated with U18666A with or without 25-HC for 12 hour and then incubated with APAP (15 mM) for 6 hours to determine cell viability by trypan blue exclusion. ( b ) Cell viability of ASMase −/− PMH after incubation with APAP (15 mM) with or without 25-HC pretreatment. ( c,d ) ASMase +/+ and ASMase −/− PMH expressing Lamp-GFP and mtKeima with or without pretreatment with U18666A or 25-HC were incubated with APAP (5 mM) for 3 hours to analyze lysosomal colocalization with mitochondria by confocal imaging. ( e,f ) 5 images per treatment of 3 different experiments were analyzed with Image J to assess the percentage of lysosomal colocalization with mitochondria. Data are expressed as mean ± SEM of 3 independent experiments. *p
    Figure Legend Snippet: Effect of U18666A and 25-HC in lysosomal colocalization with mitochondria and APAP susceptibility ( a ) ASMase +/+ PMH were pretreated with U18666A with or without 25-HC for 12 hour and then incubated with APAP (15 mM) for 6 hours to determine cell viability by trypan blue exclusion. ( b ) Cell viability of ASMase −/− PMH after incubation with APAP (15 mM) with or without 25-HC pretreatment. ( c,d ) ASMase +/+ and ASMase −/− PMH expressing Lamp-GFP and mtKeima with or without pretreatment with U18666A or 25-HC were incubated with APAP (5 mM) for 3 hours to analyze lysosomal colocalization with mitochondria by confocal imaging. ( e,f ) 5 images per treatment of 3 different experiments were analyzed with Image J to assess the percentage of lysosomal colocalization with mitochondria. Data are expressed as mean ± SEM of 3 independent experiments. *p

    Techniques Used: Incubation, Expressing, Imaging

    15) Product Images from "The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages"

    Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029042

    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 Legend Snippet: 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.

    Techniques Used: Incubation, Staining, Epifluorescence Microscopy

    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.
    Figure Legend Snippet: 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.

    Techniques Used: Incubation, Expressing

    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 Legend Snippet: 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.

    Techniques Used: Activity Assay, Incubation

    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 Legend Snippet: 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.

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    16) Product Images from "Inhibition of cholesterol recycling impairs cellular PrPSc propagation"

    Article Title: Inhibition of cholesterol recycling impairs cellular PrPSc propagation

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-009-0158-4

    U18666A treatment increases PrP Sc degradation. Upper panel ScN2a cells were treated for 1 day with U18666A (3 μg/ml) or were left untreated. Cells were either lysed after 1 day and lysates were treated with PK (lanes 1–4) or were incubated for further 2 days (day 3; lanes 5–12) with or without U18666A in presence or absence of bafilomycin A (10 nM). Then cells were lysed and subjected to PK digestion. All samples were separated by SDS-PAGE followed by immunoblot. For detection of PrP, mAb 4H11 was used. The experiment was performed in duplicate. Lower panel Samples without PK digestion were analyzed by immunoblot for total PrP using mAb 4H11 and for β-actin levels ( arrow ) for control of equal loading
    Figure Legend Snippet: U18666A treatment increases PrP Sc degradation. Upper panel ScN2a cells were treated for 1 day with U18666A (3 μg/ml) or were left untreated. Cells were either lysed after 1 day and lysates were treated with PK (lanes 1–4) or were incubated for further 2 days (day 3; lanes 5–12) with or without U18666A in presence or absence of bafilomycin A (10 nM). Then cells were lysed and subjected to PK digestion. All samples were separated by SDS-PAGE followed by immunoblot. For detection of PrP, mAb 4H11 was used. The experiment was performed in duplicate. Lower panel Samples without PK digestion were analyzed by immunoblot for total PrP using mAb 4H11 and for β-actin levels ( arrow ) for control of equal loading

    Techniques Used: Incubation, SDS Page

    Rab 9 overexpression impairs PrP Sc propagation but partially rescues the interference induced by cholesterol accumulation. ScN2a cells were transiently transfected with pEGFP-rab 9 or with pcDNA3.1 as a control. Following transfection, treatment with U18666A (3 μg/ml) was performed for 2 days. Then cells were lysed, aliquots were digested with PK, and samples were analyzed by immunoblot using mAb 4H11. A representative duplicate experiment is shown ( upper panel ). To enable better comparison, samples analyzed by one immunoblot were opposed to each other. Expression of EGFP-rab 9 was detected using a polyclonal anti-GFP antibody. PrP Sc signals of five independent experiments were evaluated densitometrically (ImageQuant TL), and statistical analysis was performed ( lower panel , ns not significant; * P
    Figure Legend Snippet: Rab 9 overexpression impairs PrP Sc propagation but partially rescues the interference induced by cholesterol accumulation. ScN2a cells were transiently transfected with pEGFP-rab 9 or with pcDNA3.1 as a control. Following transfection, treatment with U18666A (3 μg/ml) was performed for 2 days. Then cells were lysed, aliquots were digested with PK, and samples were analyzed by immunoblot using mAb 4H11. A representative duplicate experiment is shown ( upper panel ). To enable better comparison, samples analyzed by one immunoblot were opposed to each other. Expression of EGFP-rab 9 was detected using a polyclonal anti-GFP antibody. PrP Sc signals of five independent experiments were evaluated densitometrically (ImageQuant TL), and statistical analysis was performed ( lower panel , ns not significant; * P

    Techniques Used: Over Expression, Transfection, Expressing

    Dose- and time-dependent reduction of PrP Sc in persistently infected cells, but no inhibition of primary infection upon chemical induction of lysosomal cholesterol accumulation. a ScN2a cells were treated for 3 days with various concentrations (0, 1, 3, or 5 μg/ml) of U18666A as indicated. Cells were lysed, digested with PK ( +PK ) or not ( −PK ), and proteins were analyzed by immunoblot. Mab 4H11 was used for detection of PrP-specific bands. The immunoblot was dehybridized and incubated with an anti-β-actin mAb to control for equal loading ( lower panel ). b Treatment of ScN2a cells with U18666A (3 μg/ml) was performed for 3 and 7 days in parallel to mock treatment. At the different time points, U18666A-treated and mock-treated cells were lysed. Lysates were digested with PK ( +PK ) or not ( −PK ) and were subjected to immunoblot analysis using mAb 4H11. Equal loading of lysates of the different time points was confirmed by reprobing the membrane with anti-β-actin mAb ( lower panel ). c 3F4-N2a cells were left untreated or were pretreated for 4 h with U18666A (3 μg/ml) prior to prion infection. For prion infection, brain homogenates (1%) from RML- or 22L-infected terminally sick mice were added for 24 h to the culture medium. Cells that had not been pretreated were infected either in the presence (+) or absence (−) of U18666A for 24 h. For pretreated cells, the U18666A treatment was continued for the time period of infection (+4; 28 h treatment in total). After 24 h, brain homogenates and U18666A were removed, cells were washed and passaged three times 1:10. Passage 3 was lysed, lysates were treated with PK ( +PK ) or left untreated ( −PK ) and were then subjected to a solubility assay. Pellet fractions of samples + PK ( P/+PK ) and supernatant fractions of samples −PK ( S/−PK ) were analyzed by immunoblot with mAb 3F4, which selectively detects newly generated 3F4-PrP Sc in the pellet fractions (P/+PK) and 3F4-PrP c in the supernatant (S/−PK)
    Figure Legend Snippet: Dose- and time-dependent reduction of PrP Sc in persistently infected cells, but no inhibition of primary infection upon chemical induction of lysosomal cholesterol accumulation. a ScN2a cells were treated for 3 days with various concentrations (0, 1, 3, or 5 μg/ml) of U18666A as indicated. Cells were lysed, digested with PK ( +PK ) or not ( −PK ), and proteins were analyzed by immunoblot. Mab 4H11 was used for detection of PrP-specific bands. The immunoblot was dehybridized and incubated with an anti-β-actin mAb to control for equal loading ( lower panel ). b Treatment of ScN2a cells with U18666A (3 μg/ml) was performed for 3 and 7 days in parallel to mock treatment. At the different time points, U18666A-treated and mock-treated cells were lysed. Lysates were digested with PK ( +PK ) or not ( −PK ) and were subjected to immunoblot analysis using mAb 4H11. Equal loading of lysates of the different time points was confirmed by reprobing the membrane with anti-β-actin mAb ( lower panel ). c 3F4-N2a cells were left untreated or were pretreated for 4 h with U18666A (3 μg/ml) prior to prion infection. For prion infection, brain homogenates (1%) from RML- or 22L-infected terminally sick mice were added for 24 h to the culture medium. Cells that had not been pretreated were infected either in the presence (+) or absence (−) of U18666A for 24 h. For pretreated cells, the U18666A treatment was continued for the time period of infection (+4; 28 h treatment in total). After 24 h, brain homogenates and U18666A were removed, cells were washed and passaged three times 1:10. Passage 3 was lysed, lysates were treated with PK ( +PK ) or left untreated ( −PK ) and were then subjected to a solubility assay. Pellet fractions of samples + PK ( P/+PK ) and supernatant fractions of samples −PK ( S/−PK ) were analyzed by immunoblot with mAb 3F4, which selectively detects newly generated 3F4-PrP Sc in the pellet fractions (P/+PK) and 3F4-PrP c in the supernatant (S/−PK)

    Techniques Used: Infection, Inhibition, Incubation, Mouse Assay, Solubility, Generated

    Lipid raft association of PrP c is not altered in U18666A-treated cells. a N2a cells were treated for 3 days with U18666A ( right panel ) or left untreated ( left panel ) and were subjected to a flotation assay. Ten fractions from the top to the bottom of the centrifuge tube were collected and analyzed by immunoblot with mAb 4H11. b Aliquots of gradient fractions were spotted on a nitrocellulose membrane and GM1 was detected by incubation of the membrane with HRP-conjugated cholera toxin subunit B (CtxB)
    Figure Legend Snippet: Lipid raft association of PrP c is not altered in U18666A-treated cells. a N2a cells were treated for 3 days with U18666A ( right panel ) or left untreated ( left panel ) and were subjected to a flotation assay. Ten fractions from the top to the bottom of the centrifuge tube were collected and analyzed by immunoblot with mAb 4H11. b Aliquots of gradient fractions were spotted on a nitrocellulose membrane and GM1 was detected by incubation of the membrane with HRP-conjugated cholera toxin subunit B (CtxB)

    Techniques Used: Incubation

    No changes in membrane localization and turn-over of PrP c in U18666A treated N2a cells. a N2a cells were treated for 3 days with U18666A (3 μg/ml) or left untreated as indicated. For release of PrP c by PIPLC ( left panel ), cells were incubated for 4 h with or without the enzyme. Treatment with U18666A was continued during PIPLC digestion. Then cells were lysed and lysates were subjected to immunoblot analysis using mAb 4H11for detection of PrP specific bands. b N2a cells with or without U18666A treatment for 3 days were fixed, permeabilized, and stained in indirect immunofluorescence with anti-PrP mAb 4H11 and Cy2-conjugated anti-mouse IgG. c Biotinylation of N2a cells treated for 2 days with or without U18666A was performed and cultures were chased at 37°C in culture medium −/+U18666A for 60 min or were processed immediately. After 0 or 60 min chase, one culture dish each of treated or untreated cells was incubated with trypsin. PrP from cell lysates was immunoprecipitated using pAb A7, subjected to immunoblot, and detected by incubation with horseradish peroxidase (HRP)-conjugated streptavidin. A representative immunoblot of three independent experiments is shown. PrP c signals of the 60 min time point with (internalized PrP c ) or without (total PrP c ) trypsin digestion were densitometrically analyzed. The amount of internalized PrP c was expressed as a percentage of total PrP c . The lower panel depicts statistical evaluation of the difference between internalized PrP c in mock-treated or U18666A-treated cells using Student’s t -test ( ns not significant; P -value > 0.05; bars indicate standard error)
    Figure Legend Snippet: No changes in membrane localization and turn-over of PrP c in U18666A treated N2a cells. a N2a cells were treated for 3 days with U18666A (3 μg/ml) or left untreated as indicated. For release of PrP c by PIPLC ( left panel ), cells were incubated for 4 h with or without the enzyme. Treatment with U18666A was continued during PIPLC digestion. Then cells were lysed and lysates were subjected to immunoblot analysis using mAb 4H11for detection of PrP specific bands. b N2a cells with or without U18666A treatment for 3 days were fixed, permeabilized, and stained in indirect immunofluorescence with anti-PrP mAb 4H11 and Cy2-conjugated anti-mouse IgG. c Biotinylation of N2a cells treated for 2 days with or without U18666A was performed and cultures were chased at 37°C in culture medium −/+U18666A for 60 min or were processed immediately. After 0 or 60 min chase, one culture dish each of treated or untreated cells was incubated with trypsin. PrP from cell lysates was immunoprecipitated using pAb A7, subjected to immunoblot, and detected by incubation with horseradish peroxidase (HRP)-conjugated streptavidin. A representative immunoblot of three independent experiments is shown. PrP c signals of the 60 min time point with (internalized PrP c ) or without (total PrP c ) trypsin digestion were densitometrically analyzed. The amount of internalized PrP c was expressed as a percentage of total PrP c . The lower panel depicts statistical evaluation of the difference between internalized PrP c in mock-treated or U18666A-treated cells using Student’s t -test ( ns not significant; P -value > 0.05; bars indicate standard error)

    Techniques Used: Incubation, Staining, Immunofluorescence, Immunoprecipitation

    17) Product Images from "The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus"

    Article Title: The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.01053-16

    U18666A treatment inhibits HCV particle secretion but not assembly in cell culture and primary cells. HCV-replicating cells were treated with 2 μg/ml of U18666A for 16 h. (A) The amount of released viral genomes in the supernatant was determined by RT-PCR. The graph shows the data from six independent experiments, with SEMs. (B) To determine the amount of released infectious viral particles in the supernatant, a TCID 50 assay was performed. The graph shows the relative data from three independent experiments, with SEMs. (C) The amount of released viral particles in the supernatant was determined by core Ag assay. The graph shows the data from seven independent experiments, with SEMs. (D) The amount of intracellular viral particles was assayed by TCID 50 of cells lysed by freeze-thawing. The graph shows the relative data from three independent experiments, with SEMs. (E) Primary human hepatocytes were infected over night with supernatant of HCV-replicating cells. Cells were treated 56 h p.i. with 2 μg/ml of U18666A for 16 h. Viral replication and particle release were determined by RT-PCR of cell lysates and supernatants, respectively. The graph shows the relative data from three independent experiments, with SEMs. (F) Huh7.5 cells were infected for 5 h with supernatant of HCV-replicating cells. To determine the entry-inhibiting ability of U18666A, it was added at a concentration of 2 μg/ml. As positive controls for entry inhibition, the supernatant was supplemented with 1 μg/ml of anti-E2 or anti-ApoE antibody. Bound virus was removed by short trypsin treatment before analysis of entered virus by RT-PCR. The graph shows the relative data from four independent experiments, with SEMs.
    Figure Legend Snippet: U18666A treatment inhibits HCV particle secretion but not assembly in cell culture and primary cells. HCV-replicating cells were treated with 2 μg/ml of U18666A for 16 h. (A) The amount of released viral genomes in the supernatant was determined by RT-PCR. The graph shows the data from six independent experiments, with SEMs. (B) To determine the amount of released infectious viral particles in the supernatant, a TCID 50 assay was performed. The graph shows the relative data from three independent experiments, with SEMs. (C) The amount of released viral particles in the supernatant was determined by core Ag assay. The graph shows the data from seven independent experiments, with SEMs. (D) The amount of intracellular viral particles was assayed by TCID 50 of cells lysed by freeze-thawing. The graph shows the relative data from three independent experiments, with SEMs. (E) Primary human hepatocytes were infected over night with supernatant of HCV-replicating cells. Cells were treated 56 h p.i. with 2 μg/ml of U18666A for 16 h. Viral replication and particle release were determined by RT-PCR of cell lysates and supernatants, respectively. The graph shows the relative data from three independent experiments, with SEMs. (F) Huh7.5 cells were infected for 5 h with supernatant of HCV-replicating cells. To determine the entry-inhibiting ability of U18666A, it was added at a concentration of 2 μg/ml. As positive controls for entry inhibition, the supernatant was supplemented with 1 μg/ml of anti-E2 or anti-ApoE antibody. Bound virus was removed by short trypsin treatment before analysis of entered virus by RT-PCR. The graph shows the relative data from four independent experiments, with SEMs.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Infection, Concentration Assay, Inhibition

    Preferentially, HCV particles with low density are retained intracellularly. (A) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The cells were lysed by repeated freeze-thaw cycles and loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs. (B) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The supernatant was loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs.
    Figure Legend Snippet: Preferentially, HCV particles with low density are retained intracellularly. (A) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The cells were lysed by repeated freeze-thaw cycles and loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs. (B) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The supernatant was loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction

    LAMP2-positive rings are filled with lipids and could represent multilamellar bodies filled with HCV particles. (A) CLSM analysis of untreated HCV-replicating cells. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (B) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (C) CLSM analysis of HCV-replicating cells which were left untreated or treated with 2 μg/ml of U18666A for 16 h. ApoE is visualized in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. (D) TEM images of ultrathin sections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Ultrathin sections were stained with uranyl acetate to identify cellular and viral structures. (E) TEM image of cryosections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against LAMP2 or HERV-K as a negative control and stained with uranyl acetate. (F) TEM image of cryosections of Huh7.5 cells electroporated with J6 or GND which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against core and E2 and stained with uranyl acetate.
    Figure Legend Snippet: LAMP2-positive rings are filled with lipids and could represent multilamellar bodies filled with HCV particles. (A) CLSM analysis of untreated HCV-replicating cells. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (B) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (C) CLSM analysis of HCV-replicating cells which were left untreated or treated with 2 μg/ml of U18666A for 16 h. ApoE is visualized in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. (D) TEM images of ultrathin sections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Ultrathin sections were stained with uranyl acetate to identify cellular and viral structures. (E) TEM image of cryosections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against LAMP2 or HERV-K as a negative control and stained with uranyl acetate. (F) TEM image of cryosections of Huh7.5 cells electroporated with J6 or GND which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against core and E2 and stained with uranyl acetate.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Marker, Fluorescence, Transmission Electron Microscopy, Labeling, Negative Control

    U18666A has no effect on NS5A localization, but ApoE accumulates and LAMP2 forms ring-like structures. Shown are results of CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. (A) NS5A is visualized in red; nuclei were stained with DAPI, in blue, and ApoE is visualized in green. (B) The fluorescence intensity of ApoE was measured in untreated and U18666A-treated cells. The data were confirmed by quantitative Western blot analysis of cellular lysates using ApoE- and actin-specific antibodies. The quantification is based on three independent experiments. (C) Pearson's overlap coefficient of NS5A and ApoE was calculated in untreated and U18666A-treated cells. (D) The amount of secreted ApoE was measured using quantitative Western blotting of TCA-precipitated samples of untreated and U18666A-treated cells. The quantification is based on three independent experiments. (E) NS5A is visualized in red; cholesterol was stained with filipin in cyan, and the lysosomal marker LAMP2 is visualized in green. The enlarged images are shown for treated and untreated cells. (F) The fluorescence intensity of LAMP2 was measured in untreated and U18666A-treated cells. The findings were confirmed by quantitative Western blotting of cell lysates. The quantification is based on three independent experiments. (G) Huh7.5 cells expressing Rab7-YFP were stained for LAMP2, in red. Treatment of U18666A led to the formation of Rab7- and LAMP2-positive rings.
    Figure Legend Snippet: U18666A has no effect on NS5A localization, but ApoE accumulates and LAMP2 forms ring-like structures. Shown are results of CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. (A) NS5A is visualized in red; nuclei were stained with DAPI, in blue, and ApoE is visualized in green. (B) The fluorescence intensity of ApoE was measured in untreated and U18666A-treated cells. The data were confirmed by quantitative Western blot analysis of cellular lysates using ApoE- and actin-specific antibodies. The quantification is based on three independent experiments. (C) Pearson's overlap coefficient of NS5A and ApoE was calculated in untreated and U18666A-treated cells. (D) The amount of secreted ApoE was measured using quantitative Western blotting of TCA-precipitated samples of untreated and U18666A-treated cells. The quantification is based on three independent experiments. (E) NS5A is visualized in red; cholesterol was stained with filipin in cyan, and the lysosomal marker LAMP2 is visualized in green. The enlarged images are shown for treated and untreated cells. (F) The fluorescence intensity of LAMP2 was measured in untreated and U18666A-treated cells. The findings were confirmed by quantitative Western blotting of cell lysates. The quantification is based on three independent experiments. (G) Huh7.5 cells expressing Rab7-YFP were stained for LAMP2, in red. Treatment of U18666A led to the formation of Rab7- and LAMP2-positive rings.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Fluorescence, Western Blot, Marker, Expressing

    Time- and dose-dependent inhibition of viral replication by U18666A. (A) Huh9-13 cells were treated with 10 μg/ml of simvastatin or the indicated concentrations of U18666A for 72 h. w/o, without treatment. Viral replication was determined by RT-PCR. The graph shows the relative data from three independent experiments, with SEMs. (B) Huh7.5 cells were transfected with the J6-Luc construct and treated with 1,000 U of interferon alpha, 10 μg/ml of simvastatin, or the indicated concentrations of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay. The graph shows the relative data from three independent experiments, with SEMs. (C) Huh7.5 cells were transfected with the J6-Luc construct and treated with 2 μg/ml of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay at different time points. The graph shows the relative data from three independent experiments, with SEMs. (D) Huh9-13 and Huh7.5 J6 cells were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. Viral replication was determined by RT-PCR. The graph shows the relative data from at least three independent experiments, with SEMs. ns, not significant. (E) Western blot analysis of cellular lysates derived from Huh9-13 and Huh7.5 J6 cells which were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. For detection, specific antibodies against NS3, NS5A, and core were used. Detection of β-actin served as a loading control. (F) Densitometric quantification of Western blots shown in panel E. The graphs show the quantification of at least three independent experiments, with SEMs.
    Figure Legend Snippet: Time- and dose-dependent inhibition of viral replication by U18666A. (A) Huh9-13 cells were treated with 10 μg/ml of simvastatin or the indicated concentrations of U18666A for 72 h. w/o, without treatment. Viral replication was determined by RT-PCR. The graph shows the relative data from three independent experiments, with SEMs. (B) Huh7.5 cells were transfected with the J6-Luc construct and treated with 1,000 U of interferon alpha, 10 μg/ml of simvastatin, or the indicated concentrations of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay. The graph shows the relative data from three independent experiments, with SEMs. (C) Huh7.5 cells were transfected with the J6-Luc construct and treated with 2 μg/ml of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay at different time points. The graph shows the relative data from three independent experiments, with SEMs. (D) Huh9-13 and Huh7.5 J6 cells were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. Viral replication was determined by RT-PCR. The graph shows the relative data from at least three independent experiments, with SEMs. ns, not significant. (E) Western blot analysis of cellular lysates derived from Huh9-13 and Huh7.5 J6 cells which were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. For detection, specific antibodies against NS3, NS5A, and core were used. Detection of β-actin served as a loading control. (F) Densitometric quantification of Western blots shown in panel E. The graphs show the quantification of at least three independent experiments, with SEMs.

    Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Luciferase, Reporter Gene Assay, Western Blot, Derivative Assay

    U18666A treatment leads to the formation of nonfunctional lysosomal MLBs in which the HCV particles accumulate. The viral RNA is transported from the replication complex (RC) to the assembly complex (AC) at the membranous web, where the particles are formed. VLDLs as well as LVPs reach the MVBs, where they are sorted for either release or degradation by fusion with lysosomes. Addition of U18666A disturbs this mechanism by blocking the secretion of cholesterol and exosomes by MVBs. This leads to an accumulation of these HCV particles determined for release in exosomes. As the endolysosomes are overloaded with cholesterol and other lipids, they form nonfunctional MLBs. The HCV particles that are determined for lysosomal degradation accumulate here and are prevented from degradation due to the dysfunctionality of the MLBs.
    Figure Legend Snippet: U18666A treatment leads to the formation of nonfunctional lysosomal MLBs in which the HCV particles accumulate. The viral RNA is transported from the replication complex (RC) to the assembly complex (AC) at the membranous web, where the particles are formed. VLDLs as well as LVPs reach the MVBs, where they are sorted for either release or degradation by fusion with lysosomes. Addition of U18666A disturbs this mechanism by blocking the secretion of cholesterol and exosomes by MVBs. This leads to an accumulation of these HCV particles determined for release in exosomes. As the endolysosomes are overloaded with cholesterol and other lipids, they form nonfunctional MLBs. The HCV particles that are determined for lysosomal degradation accumulate here and are prevented from degradation due to the dysfunctionality of the MLBs.

    Techniques Used: Blocking Assay

    HCV structural proteins accumulate intracellularly in endosomal structures. (A) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. HCV core is visualized in green; the nuclei were stained with DAPI, in blue, and the exosomal marker CD63 is visualized in red. Shown are the fluorescence intensities of DAPI, core, and CD63 along the white arrows. Enlarged images are shown for treated and untreated cells. (B) The fluorescence intensities of core and CD63 were measured in untreated and U18666A-treated cells (left). The amount of CD63 was also determined by quantitative Western blotting using cell lysates (right). The quantification is based on three independent experiments. (C) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml ofU18666A for 16 h. HCV core is visualized in red; the nuclei were stained with DAPI, in blue, and the autophagosomal marker LC3 is visualized in green. The graphs show the fluorescence intensities of DAPI, core, and LC3 along the white arrows in the zoomed image. The enlarged images are shown for treated and untreated cells. (D) The weighted colocalization coefficients of core and CD63 were calculated in untreated and U18666A-treated cells. (E) CLSM analysis of Huh7.5 cells that were electroporated with HCV J6 RNA and plasmids encoding the indicated fusion proteins. HCV core was costained with a specific antibody. (F) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. Huh7.5 cells were transfected with an HCV construct harboring an mCherry fusion tag inside the hypervariable region (HVR) of E1, which is visualized in red. The nuclei were stained with DAPI, in blue, and the lysosomal marker LAMP2 is visualized in green. (G) Zoomed image of a part of panel F with intensity profiles showing the fluorescence intensities of DAPI, LAMP2, and E1 along the white arrows.
    Figure Legend Snippet: HCV structural proteins accumulate intracellularly in endosomal structures. (A) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. HCV core is visualized in green; the nuclei were stained with DAPI, in blue, and the exosomal marker CD63 is visualized in red. Shown are the fluorescence intensities of DAPI, core, and CD63 along the white arrows. Enlarged images are shown for treated and untreated cells. (B) The fluorescence intensities of core and CD63 were measured in untreated and U18666A-treated cells (left). The amount of CD63 was also determined by quantitative Western blotting using cell lysates (right). The quantification is based on three independent experiments. (C) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml ofU18666A for 16 h. HCV core is visualized in red; the nuclei were stained with DAPI, in blue, and the autophagosomal marker LC3 is visualized in green. The graphs show the fluorescence intensities of DAPI, core, and LC3 along the white arrows in the zoomed image. The enlarged images are shown for treated and untreated cells. (D) The weighted colocalization coefficients of core and CD63 were calculated in untreated and U18666A-treated cells. (E) CLSM analysis of Huh7.5 cells that were electroporated with HCV J6 RNA and plasmids encoding the indicated fusion proteins. HCV core was costained with a specific antibody. (F) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. Huh7.5 cells were transfected with an HCV construct harboring an mCherry fusion tag inside the hypervariable region (HVR) of E1, which is visualized in red. The nuclei were stained with DAPI, in blue, and the lysosomal marker LAMP2 is visualized in green. (G) Zoomed image of a part of panel F with intensity profiles showing the fluorescence intensities of DAPI, LAMP2, and E1 along the white arrows.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Marker, Fluorescence, Western Blot, Transfection, Construct

    18) Product Images from "Proteome-wide Dysregulation by PRA1 Depletion Delineates a Role of PRA1 in Lipid Transport and Cell Migration *"

    Article Title: Proteome-wide Dysregulation by PRA1 Depletion Delineates a Role of PRA1 in Lipid Transport and Cell Migration *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M900641-MCP200

    U18666A treatment phenocopied the effect of PRA1 knockdown in NPC cells. A , Cellular cholesterol accumulation in PRA1-knockdown cells. Cellular cholesterols in PRA1-knockdown (K3–2) or control (C15–3) cells were visualized by filipin staining
    Figure Legend Snippet: U18666A treatment phenocopied the effect of PRA1 knockdown in NPC cells. A , Cellular cholesterol accumulation in PRA1-knockdown cells. Cellular cholesterols in PRA1-knockdown (K3–2) or control (C15–3) cells were visualized by filipin staining

    Techniques Used: Staining

    A , A model depicting the role of PRA1 in morphogenesis of NPC cells. PRA1 knockdown affects cellular cholesterol transport, thereby causing cellular cholesterol accumulation reminiscent of the effect of U18666A. This cholesterol accumulation elicits adaptive
    Figure Legend Snippet: A , A model depicting the role of PRA1 in morphogenesis of NPC cells. PRA1 knockdown affects cellular cholesterol transport, thereby causing cellular cholesterol accumulation reminiscent of the effect of U18666A. This cholesterol accumulation elicits adaptive

    Techniques Used:

    19) Product Images from "Non-esterified Cholesterol Content of Lysosomes Modulates Susceptibility to Oxidant-induced Permeabilization"

    Article Title: Non-esterified Cholesterol Content of Lysosomes Modulates Susceptibility to Oxidant-induced Permeabilization

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2010.11.006

    U18666A-, imipramine- and clozapine-mediated inhibition of apoptosis initiated by photoirradiation of NPe6-sensitized cultures. 1c1c7 cultures were treated with nothing (NT) or different concentrations of U18666A (UA), imipramine (IPM), or clozapine (CZP)
    Figure Legend Snippet: U18666A-, imipramine- and clozapine-mediated inhibition of apoptosis initiated by photoirradiation of NPe6-sensitized cultures. 1c1c7 cultures were treated with nothing (NT) or different concentrations of U18666A (UA), imipramine (IPM), or clozapine (CZP)

    Techniques Used: Inhibition

    U18666A-mediated suppression of H 2 O 2 -induced LMP and DEVDase activation. (A) 1c1c7 cultures were incubated with 100, 150 or 200 µM H 2 O 2 for different lengths of time prior to being harvested for subsequent analyses of DEVDase activities. (B) 1c1c7
    Figure Legend Snippet: U18666A-mediated suppression of H 2 O 2 -induced LMP and DEVDase activation. (A) 1c1c7 cultures were incubated with 100, 150 or 200 µM H 2 O 2 for different lengths of time prior to being harvested for subsequent analyses of DEVDase activities. (B) 1c1c7

    Techniques Used: Activation Assay, Incubation

    PDT-induced oxidation of lysosomal cholesterol. (A) 1c1c7 cultures were not treated (NT) or exposed to 0.1 or 0.75 µM U18666A (UA) for ~20 h prior to being washed and refed with medium containing 33 µM NPe6. After 2 h of sensitization
    Figure Legend Snippet: PDT-induced oxidation of lysosomal cholesterol. (A) 1c1c7 cultures were not treated (NT) or exposed to 0.1 or 0.75 µM U18666A (UA) for ~20 h prior to being washed and refed with medium containing 33 µM NPe6. After 2 h of sensitization

    Techniques Used:

    U18666A does not suppress the pro-apoptotic activities of agents that do not induce LMP. 1c1c7 cultures were treated with nothing, 1 µM U18666A (UA), or 12.5 µM imipramine (IPM) for ~20 h. Some cultures were subsequently treated with 25
    Figure Legend Snippet: U18666A does not suppress the pro-apoptotic activities of agents that do not induce LMP. 1c1c7 cultures were treated with nothing, 1 µM U18666A (UA), or 12.5 µM imipramine (IPM) for ~20 h. Some cultures were subsequently treated with 25

    Techniques Used:

    U18666A-induced endosomal/ysosomal accumulation of non-esterified sterols. (A) 1c1c7 cultures were incubated with dextran-10000 tetramethylrhodamine (D10K-TMR) overnight prior to being washed and refed with fresh medium. At the time of refeeding some
    Figure Legend Snippet: U18666A-induced endosomal/ysosomal accumulation of non-esterified sterols. (A) 1c1c7 cultures were incubated with dextran-10000 tetramethylrhodamine (D10K-TMR) overnight prior to being washed and refed with fresh medium. At the time of refeeding some

    Techniques Used: Incubation

    CAD suppression of LMP. (A) 1c1c7 cultures were treated with nothing, 1 µM U18666A (UA), or 12.5 µM imipramine (IPM) for ~22 h prior to being washed and refed with fresh medium (± 33 µM NPe6 for 1 h) and subsequently photoirradiated
    Figure Legend Snippet: CAD suppression of LMP. (A) 1c1c7 cultures were treated with nothing, 1 µM U18666A (UA), or 12.5 µM imipramine (IPM) for ~22 h prior to being washed and refed with fresh medium (± 33 µM NPe6 for 1 h) and subsequently photoirradiated

    Techniques Used:

    Time-dependent recovery of sensitivity to PDT-induced LMP following washout of U18666A. (A) 1c1c7 cultures were treated with nothing (NT) or 1 µM U18666A for 24 h prior to being washed and refed with new medium lacking U18666A. Cultures were stained
    Figure Legend Snippet: Time-dependent recovery of sensitivity to PDT-induced LMP following washout of U18666A. (A) 1c1c7 cultures were treated with nothing (NT) or 1 µM U18666A for 24 h prior to being washed and refed with new medium lacking U18666A. Cultures were stained

    Techniques Used: Staining

    U18666A suppresses PDT-induced oxidation of C11-BODIPY. (A) 1c1c7 cultures were exposed to 4 µM C11-BODIPY (C11) for 1 h prior to replacing medium and examination by fluorescence microscopy for the distribution of reduced (red) and oxidized (green)
    Figure Legend Snippet: U18666A suppresses PDT-induced oxidation of C11-BODIPY. (A) 1c1c7 cultures were exposed to 4 µM C11-BODIPY (C11) for 1 h prior to replacing medium and examination by fluorescence microscopy for the distribution of reduced (red) and oxidized (green)

    Techniques Used: Fluorescence, Microscopy

    Effects of U18666A, imipramine and clozapine on 1c1c7 cell growth and filipin staining. (A) Cultures of murine hepatoma 1c1c7 cells were treated with the indicated concentrations of U18666A, imipramine, or clozapine before being harvested and counted.
    Figure Legend Snippet: Effects of U18666A, imipramine and clozapine on 1c1c7 cell growth and filipin staining. (A) Cultures of murine hepatoma 1c1c7 cells were treated with the indicated concentrations of U18666A, imipramine, or clozapine before being harvested and counted.

    Techniques Used: Staining

    20) Product Images from "Aberrant DR5 transport through disruption of lysosomal function suggests a novel mechanism for receptor activation"

    Article Title: Aberrant DR5 transport through disruption of lysosomal function suggests a novel mechanism for receptor activation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11073

    Free cholesterol is important for DR5 trafficking and 5-FU-induced apoptosis Filipin III, a fluorescent polyene macrolide antibiotic, was used to localize unesterified cholesterol in control, 5-FU (768 μM), Baf A (100 nM), CQ (20 μM) and U18666A (5 μg/mL)-treated and subsequently paraformaldehyde fixed HCT116 wt cells A. HCT116 control cells and cells induced with 5-FU, U18666A or their combination for 20 h were fixed in 3.8% formaldehyde for 20 min and exposed to a DR5 specific antibody (F2/B4, green). Before fixation, cells were incubated in the presence of lysotracker red (red, 100 nM) for 1 h. B. Cell nuclei were visualized by Hoechst 33342. Bars, 10 μm p53, cleaved PARP, lamin A (cle PARP, cle lamin A) and processing of caspase-8 were analyzed by immunoblotting of SDS-PAGE-separated cell lysates harvested 24 h post-treatment using either 5-FU (768 μM) alone, or in combination with U18666A (0.1-5 μg/mL) C. or MβCD (2.5 and 5 mM). D. GAPDH (C) and α-tubulin (D) served as a marker of equal sample loading. Processed caspase-8 fragments and the short isoform of DR5 are indicated by asterisks (C and D). An additional DR5-related fragment appearing in 5-FU and U18666A as well as 5-FU and MβCD co-treated cells is indicated by an arrow (C and D).
    Figure Legend Snippet: Free cholesterol is important for DR5 trafficking and 5-FU-induced apoptosis Filipin III, a fluorescent polyene macrolide antibiotic, was used to localize unesterified cholesterol in control, 5-FU (768 μM), Baf A (100 nM), CQ (20 μM) and U18666A (5 μg/mL)-treated and subsequently paraformaldehyde fixed HCT116 wt cells A. HCT116 control cells and cells induced with 5-FU, U18666A or their combination for 20 h were fixed in 3.8% formaldehyde for 20 min and exposed to a DR5 specific antibody (F2/B4, green). Before fixation, cells were incubated in the presence of lysotracker red (red, 100 nM) for 1 h. B. Cell nuclei were visualized by Hoechst 33342. Bars, 10 μm p53, cleaved PARP, lamin A (cle PARP, cle lamin A) and processing of caspase-8 were analyzed by immunoblotting of SDS-PAGE-separated cell lysates harvested 24 h post-treatment using either 5-FU (768 μM) alone, or in combination with U18666A (0.1-5 μg/mL) C. or MβCD (2.5 and 5 mM). D. GAPDH (C) and α-tubulin (D) served as a marker of equal sample loading. Processed caspase-8 fragments and the short isoform of DR5 are indicated by asterisks (C and D). An additional DR5-related fragment appearing in 5-FU and U18666A as well as 5-FU and MβCD co-treated cells is indicated by an arrow (C and D).

    Techniques Used: Incubation, SDS Page, Marker

    21) Product Images from "Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway"

    Article Title: Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

    Journal: mBio

    doi: 10.1128/mBio.01857-15

    Lipid mixing requires EBOV GP-NPC1 interaction. (A) Lipid mixing by EBOV GPΔMuc-pseudotyped virus is significantly reduced in U2OS cells lacking the NPC1 cholesterol transporter. Asterisks indicate that values are significantly lower than those seen with wt U2OS. (B) A GP mutant unable to bind NPC1 displays impaired lipid mixing activity and infectivity. Asterisks indicate that values are significantly lower than those seen with VSV-EBOV GPΔMuc. (C) Lipid mixing is decreased by addition of the drug U18666A (10 µM), which inhibits cholesterol transport from within endosomes and induces an NPC1 disease phenotype. Asterisks indicate that values are significantly lower than those seen with the untreated control.
    Figure Legend Snippet: Lipid mixing requires EBOV GP-NPC1 interaction. (A) Lipid mixing by EBOV GPΔMuc-pseudotyped virus is significantly reduced in U2OS cells lacking the NPC1 cholesterol transporter. Asterisks indicate that values are significantly lower than those seen with wt U2OS. (B) A GP mutant unable to bind NPC1 displays impaired lipid mixing activity and infectivity. Asterisks indicate that values are significantly lower than those seen with VSV-EBOV GPΔMuc. (C) Lipid mixing is decreased by addition of the drug U18666A (10 µM), which inhibits cholesterol transport from within endosomes and induces an NPC1 disease phenotype. Asterisks indicate that values are significantly lower than those seen with the untreated control.

    Techniques Used: Mutagenesis, Activity Assay, Infection

    22) Product Images from "Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration *"

    Article Title: Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.683557

    Accumulation of LE cholesterol reduces the association of Stx6 with the TGN. A and B , CHO-WT and CHO-A6 cells treated with 3 μg/ml U18666A ( A ) or 0–4.5 μg/ml U18666A for 16 h ( B ) were stained with anti-Stx6 and filipin. Insets
    Figure Legend Snippet: Accumulation of LE cholesterol reduces the association of Stx6 with the TGN. A and B , CHO-WT and CHO-A6 cells treated with 3 μg/ml U18666A ( A ) or 0–4.5 μg/ml U18666A for 16 h ( B ) were stained with anti-Stx6 and filipin. Insets

    Techniques Used: Staining

    23) Product Images from "Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling"

    Article Title: Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling

    Journal: Glycobiology

    doi: 10.1093/glycob/cwv084

    Statins increase GlcCer and Lc3Cer in A431 cells. ( A ) Total GSL extracts from 10 6 A431G cells cultured with Crestor™ pill extract (theoretical rosuvastatin concentration 20 µM) or 5 µM lovastatin for 72 h were separated by TLC in solvent C and detected by orcinol spray for carbohydrate. Migration of standards is indicated; GlcCer, lactosylceramide, Gb 3 and Gb 4 (globo-series GSLs, globotriaosyl or tetraosylceramide, respectively), GM3 (monosialoganglioside). Statins substantially increase GlcCer and a ceramide trihexoside. ( B ) Neutral GSLs from A431S cells grown ± 10 µM lovastatin or U18666A for 48 h were separated in solvent B and detected with orcinol. GSL changes induced by U18666A are distinct from statin effects. ( C ) The TLC trihexoside region is shown; A431S cells were treated with lovastatin or vehicle control as in (B), then TLC plates were probed with anti-Lc 3 Cer, anti-Gg 3 , or VT1 B-subunit for Gb 3 . Antibody–ligand binding to standard GSLs is shown left. ( D ) MALDI MS of neutral GSLs of A431S cells treated as in (B). The ceramide trihexoside (Gb 3 ) C24:1/24:0 fatty acid region is shown, and sodiated peaks for C24:0 ( m / z 1158.8) and C24:1 (1156.8) are labeled. On lovastatin treatment, the sodiated C24:0/C24:1 peaks of Lc3Cer are apparent ( m / z 1199.8/1197.9, respectively). ( E ) TLC analysis of cholesterol and phospholipid fractions of A431S cells treated as in (B). Lipids were identified by co-migration with standards. Cholesterol from 2 × 10 5 cells was run in solvent D and detected with FeCl 3 spray; PE, PC, and SM from 5 × 10 5 cells run in solvent B were detected by iodine vapor. This figure is available in black and white in print and in colour at Glycobiology online.
    Figure Legend Snippet: Statins increase GlcCer and Lc3Cer in A431 cells. ( A ) Total GSL extracts from 10 6 A431G cells cultured with Crestor™ pill extract (theoretical rosuvastatin concentration 20 µM) or 5 µM lovastatin for 72 h were separated by TLC in solvent C and detected by orcinol spray for carbohydrate. Migration of standards is indicated; GlcCer, lactosylceramide, Gb 3 and Gb 4 (globo-series GSLs, globotriaosyl or tetraosylceramide, respectively), GM3 (monosialoganglioside). Statins substantially increase GlcCer and a ceramide trihexoside. ( B ) Neutral GSLs from A431S cells grown ± 10 µM lovastatin or U18666A for 48 h were separated in solvent B and detected with orcinol. GSL changes induced by U18666A are distinct from statin effects. ( C ) The TLC trihexoside region is shown; A431S cells were treated with lovastatin or vehicle control as in (B), then TLC plates were probed with anti-Lc 3 Cer, anti-Gg 3 , or VT1 B-subunit for Gb 3 . Antibody–ligand binding to standard GSLs is shown left. ( D ) MALDI MS of neutral GSLs of A431S cells treated as in (B). The ceramide trihexoside (Gb 3 ) C24:1/24:0 fatty acid region is shown, and sodiated peaks for C24:0 ( m / z 1158.8) and C24:1 (1156.8) are labeled. On lovastatin treatment, the sodiated C24:0/C24:1 peaks of Lc3Cer are apparent ( m / z 1199.8/1197.9, respectively). ( E ) TLC analysis of cholesterol and phospholipid fractions of A431S cells treated as in (B). Lipids were identified by co-migration with standards. Cholesterol from 2 × 10 5 cells was run in solvent D and detected with FeCl 3 spray; PE, PC, and SM from 5 × 10 5 cells run in solvent B were detected by iodine vapor. This figure is available in black and white in print and in colour at Glycobiology online.

    Techniques Used: Cell Culture, Concentration Assay, Thin Layer Chromatography, Migration, Ligand Binding Assay, Mass Spectrometry, Labeling

    24) Product Images from "Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation"

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709057

    Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.
    Figure Legend Snippet: Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.

    Techniques Used: Activation Assay, Incubation, Infection, Transfection

    Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P
    Figure Legend Snippet: Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P

    Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P
    Figure Legend Snippet: Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P

    Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Microscopy

    ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P
    Figure Legend Snippet: ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P

    Techniques Used: Labeling, Staining, Transfection

    25) Product Images from "Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes *"

    Article Title: Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.014993

    Treatment with the amino steroid U18666A of HeLa cells stably expressing SLP-1-GFP results in the formation of large, cholesterol-rich vesicles. A , upper panel , a mixture of control and SLP-1-GFP expressing HeLa cells was cultivated in the absence of
    Figure Legend Snippet: Treatment with the amino steroid U18666A of HeLa cells stably expressing SLP-1-GFP results in the formation of large, cholesterol-rich vesicles. A , upper panel , a mixture of control and SLP-1-GFP expressing HeLa cells was cultivated in the absence of

    Techniques Used: Stable Transfection, Expressing

    Reduced LAMP-2 expression on the large, cholesterol-rich SLP-1-GFP-positive vesicles. Cells stably expressing either SLP-1-GFP or SLP-1-(1–288)-GFP were treated with 3 μg/ml U18666A for 24 h. The cells were fixed and stained with filipin
    Figure Legend Snippet: Reduced LAMP-2 expression on the large, cholesterol-rich SLP-1-GFP-positive vesicles. Cells stably expressing either SLP-1-GFP or SLP-1-(1–288)-GFP were treated with 3 μg/ml U18666A for 24 h. The cells were fixed and stained with filipin

    Techniques Used: Expressing, Stable Transfection, Staining

    26) Product Images from "Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection"

    Article Title: Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056265

    CADs do not disrupt the interaction of 19 kDa GP and NPC1. (A) NPC1-FLAG-enriched LE/Lys membranes from CHO NPC1 cells were disrupted and then incubated with inhibitors for 30 min at RT: mock (4% DMSO), E64d (10 µM), compound 3.47 (13 µM), clomiphene (242 µM), Ro 48-8071 (174 µM), and U18666A (800 µM); each inhibitor was used at a concentration 100 fold over its IC 50 for inhibition of infection. The samples were then incubated with 3 µg uncleaved (GP) or cleaved (GP 19 kDa ) EBOV GP ectodomains for 1 hr at RT. Samples were then lysed, and incubated overnight with anti-FLAG beads. Bound NPC1 and GP were then eluted from beads, and run on an SDS-PAGE gel. The gel was then transferred, blotted for both NPC1 and EBOV GP, and imaged for fluorescent signal. As predicted, uncleaved GP (∼130 kDa) did not co-precipitate with NPC1 [20] , [24] . (B) The intensities of the GP, GP 19 kDa , and NPC1 bands from each sample of the blot shown in Fig. 7A were quantified and GP or GP 19 kDa was normalized to its respective NPC1 band signal. The experiment was conducted four times with similar results, and a representative experiment is shown.
    Figure Legend Snippet: CADs do not disrupt the interaction of 19 kDa GP and NPC1. (A) NPC1-FLAG-enriched LE/Lys membranes from CHO NPC1 cells were disrupted and then incubated with inhibitors for 30 min at RT: mock (4% DMSO), E64d (10 µM), compound 3.47 (13 µM), clomiphene (242 µM), Ro 48-8071 (174 µM), and U18666A (800 µM); each inhibitor was used at a concentration 100 fold over its IC 50 for inhibition of infection. The samples were then incubated with 3 µg uncleaved (GP) or cleaved (GP 19 kDa ) EBOV GP ectodomains for 1 hr at RT. Samples were then lysed, and incubated overnight with anti-FLAG beads. Bound NPC1 and GP were then eluted from beads, and run on an SDS-PAGE gel. The gel was then transferred, blotted for both NPC1 and EBOV GP, and imaged for fluorescent signal. As predicted, uncleaved GP (∼130 kDa) did not co-precipitate with NPC1 [20] , [24] . (B) The intensities of the GP, GP 19 kDa , and NPC1 bands from each sample of the blot shown in Fig. 7A were quantified and GP or GP 19 kDa was normalized to its respective NPC1 band signal. The experiment was conducted four times with similar results, and a representative experiment is shown.

    Techniques Used: Incubation, Concentration Assay, Inhibition, Infection, SDS Page

    Effects of sterol pathway inhibitors on EBOV VLP entry. SNB19 cells were pretreated with inhibitor for 1 hr, and EBOV VLP-GPΔ was then bound by spinfection at 4°C for 1 hr. Cells were then washed in media with inhibitor, incubated at 37°C for 3 hr (in inhibitor), and then processed for VLP entry as described in the Materials and Methods section. Compounds were tested in multiple (n) experiments (each in triplicate) at either the highest concentration under which no toxicity was observed (for compounds that did not inhibit infection), or at the concentration that resulted in maximum inhibition of EBOV infection ( Fig. 1 ) with minimal toxicity: SR 12813 (5 µM), (n = 4), colestolone (10 µM), (n = 5), alendronate (20 µM), (n = 3), BM 15766 (2 µM), (n = 4), amorolfine (6 µM), (n = 4), AY-9944 (5 µM), (n = 3), clomiphene (5 µM), (n = 9), Ro 48-8071 (5 µM, (n = 6), U18666A (5 µM), (n = 7), terconazole (10 µM), (n = 4), and triparanol (5 µM), (n = 3). Error bars represent standard error: * (P
    Figure Legend Snippet: Effects of sterol pathway inhibitors on EBOV VLP entry. SNB19 cells were pretreated with inhibitor for 1 hr, and EBOV VLP-GPΔ was then bound by spinfection at 4°C for 1 hr. Cells were then washed in media with inhibitor, incubated at 37°C for 3 hr (in inhibitor), and then processed for VLP entry as described in the Materials and Methods section. Compounds were tested in multiple (n) experiments (each in triplicate) at either the highest concentration under which no toxicity was observed (for compounds that did not inhibit infection), or at the concentration that resulted in maximum inhibition of EBOV infection ( Fig. 1 ) with minimal toxicity: SR 12813 (5 µM), (n = 4), colestolone (10 µM), (n = 5), alendronate (20 µM), (n = 3), BM 15766 (2 µM), (n = 4), amorolfine (6 µM), (n = 4), AY-9944 (5 µM), (n = 3), clomiphene (5 µM), (n = 9), Ro 48-8071 (5 µM, (n = 6), U18666A (5 µM), (n = 7), terconazole (10 µM), (n = 4), and triparanol (5 µM), (n = 3). Error bars represent standard error: * (P

    Techniques Used: Incubation, Concentration Assay, Infection, Inhibition

    CADs inhibit EBOV GP-mediated infection in an NPC1-dependent manner. Parental CHO cells (−−−−) and stably overexpressing CHO NPC1 cells (− − −) were pre-treated with the indicated concentration of inhibitor for 1 hr at 37°C, and then infected with VSV-GPΔ for 18 hr in the continued presence of inhibitor. Each concentration of inhibitor was tested (in duplicate) in the following number of experiments: E64d (n = 2), compound 3.47 (n = 2), clomiphene (n = 3), Ro 48-8071 (n = 3), and U18666A (n = 4). Infection values were normalized to DMSO treated samples and averaged across experiments. Error bars represent standard error.
    Figure Legend Snippet: CADs inhibit EBOV GP-mediated infection in an NPC1-dependent manner. Parental CHO cells (−−−−) and stably overexpressing CHO NPC1 cells (− − −) were pre-treated with the indicated concentration of inhibitor for 1 hr at 37°C, and then infected with VSV-GPΔ for 18 hr in the continued presence of inhibitor. Each concentration of inhibitor was tested (in duplicate) in the following number of experiments: E64d (n = 2), compound 3.47 (n = 2), clomiphene (n = 3), Ro 48-8071 (n = 3), and U18666A (n = 4). Infection values were normalized to DMSO treated samples and averaged across experiments. Error bars represent standard error.

    Techniques Used: Infection, Stable Transfection, Concentration Assay

    U18666A inhibits EBOV entry at a post internalization step and does not inhibit endosome acidification or cathepsin levels. Effects of U18666A (indicated concentration in A, 5 µM in B-E) on: (A) VLP-GPΔ and VLP-G entry; one representative of two experiments (done in triplicate). (B) VLP-GPΔ and VLP-LCMV entry; one representative of three experiments (done in duplicate). (C) VLP-GPΔ internalization and entry (controls as in Fig. 3C ); one representative of two experiments (done in triplicate). (D) Endosomal pH detected by Lysotracker Red; 10 mM NH 4 Cl was used as the control for pH neutralization; representative images from multiple coverslips from a single experiment (E) Cathepsin B, L, and combined B/L activity; E64d was used as the positive control as in Fig. 3E ; results from a single experiment performed in duplicate. In all assays, SNB19 cells were pre-treated with the indicated concentration of inhibitor for 1 hr at 37°C, and inhibitors were maintained throughout the assays. Error bars represent standard deviation from the mean of mock-treated samples: * (P
    Figure Legend Snippet: U18666A inhibits EBOV entry at a post internalization step and does not inhibit endosome acidification or cathepsin levels. Effects of U18666A (indicated concentration in A, 5 µM in B-E) on: (A) VLP-GPΔ and VLP-G entry; one representative of two experiments (done in triplicate). (B) VLP-GPΔ and VLP-LCMV entry; one representative of three experiments (done in duplicate). (C) VLP-GPΔ internalization and entry (controls as in Fig. 3C ); one representative of two experiments (done in triplicate). (D) Endosomal pH detected by Lysotracker Red; 10 mM NH 4 Cl was used as the control for pH neutralization; representative images from multiple coverslips from a single experiment (E) Cathepsin B, L, and combined B/L activity; E64d was used as the positive control as in Fig. 3E ; results from a single experiment performed in duplicate. In all assays, SNB19 cells were pre-treated with the indicated concentration of inhibitor for 1 hr at 37°C, and inhibitors were maintained throughout the assays. Error bars represent standard deviation from the mean of mock-treated samples: * (P

    Techniques Used: Concentration Assay, Neutralization, Activity Assay, Positive Control, Standard Deviation

    27) Product Images from "The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells"

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08332-9

    Interleukin (IL-10) regulation in T-helper type 1 (Th1) switching cells is not dependent on isoprenylation, vitamin D3 or cellular cholesterol content. Purified human CD4 + T cells stimulated in vitro with plate-bound α-CD3 (2 μgml −1 ) + α-CD46 (5 μgml −1 ) and recombinant human interleukin-2 (rhIL-2) (50Uml −1 ) were cultured for 36 h in the presence of selected metabolites and inhibitors as indicated. a Representative flow cytometric analysis of intracellular interferon-γ (IFNγ) and IL-10 staining of cells treated with different formulations of statin (data representative of three independent donors). b Normalised frequency of IL-10 + cells cultured in the presence of inhibitors for farnesyl-PP transferase (FTase I inhibitor) (left) ( n = 4), geranylgenaryl transferase I (GGTI-298) (centre) ( n = 4) and Rab geranylgeranyl transferase (psoromic acid) (right) ( n = 5). c Normalised frequency of IL-10 + cells cultured with 5μM atorvastatin (AT) and increasing concentrations of farnesylpyrophosphate (FPP) (left) and geranylgeranylphyrophosphate (GGPP) (right) and mevalonic acid (MA) as control ( n = 5). d Normalised frequency of IL-10 + cells cultured in the presence of increasing concentrations of U18666A ( n = 6). e Normalised frequency of IL-10 + cells cultured with 5 μM atorvastatin (AT) and increasing concentrations of calcitriol and mevalonic acid (MA) as a control ( n = 5). f Normalised total cellular cholesterol content measured by liquid chromatography mass spectrometry. The [cholesterol –H 2 O + H] + ion was the dominant adduct for cholesterol and relative intensity is shown for this ion ( n = 3). Graphs show independent donors (dots) normalised to 0μM dose of atorvastatin; bars represent median values. *
    Figure Legend Snippet: Interleukin (IL-10) regulation in T-helper type 1 (Th1) switching cells is not dependent on isoprenylation, vitamin D3 or cellular cholesterol content. Purified human CD4 + T cells stimulated in vitro with plate-bound α-CD3 (2 μgml −1 ) + α-CD46 (5 μgml −1 ) and recombinant human interleukin-2 (rhIL-2) (50Uml −1 ) were cultured for 36 h in the presence of selected metabolites and inhibitors as indicated. a Representative flow cytometric analysis of intracellular interferon-γ (IFNγ) and IL-10 staining of cells treated with different formulations of statin (data representative of three independent donors). b Normalised frequency of IL-10 + cells cultured in the presence of inhibitors for farnesyl-PP transferase (FTase I inhibitor) (left) ( n = 4), geranylgenaryl transferase I (GGTI-298) (centre) ( n = 4) and Rab geranylgeranyl transferase (psoromic acid) (right) ( n = 5). c Normalised frequency of IL-10 + cells cultured with 5μM atorvastatin (AT) and increasing concentrations of farnesylpyrophosphate (FPP) (left) and geranylgeranylphyrophosphate (GGPP) (right) and mevalonic acid (MA) as control ( n = 5). d Normalised frequency of IL-10 + cells cultured in the presence of increasing concentrations of U18666A ( n = 6). e Normalised frequency of IL-10 + cells cultured with 5 μM atorvastatin (AT) and increasing concentrations of calcitriol and mevalonic acid (MA) as a control ( n = 5). f Normalised total cellular cholesterol content measured by liquid chromatography mass spectrometry. The [cholesterol –H 2 O + H] + ion was the dominant adduct for cholesterol and relative intensity is shown for this ion ( n = 3). Graphs show independent donors (dots) normalised to 0μM dose of atorvastatin; bars represent median values. *

    Techniques Used: Purification, In Vitro, Recombinant, Cell Culture, Flow Cytometry, Staining, Liquid Chromatography, Mass Spectrometry

    28) Product Images from "Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases [S]"

    Article Title: Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M064923

    GlcChol abnormalities in NPC. A: GlcChol (nanomoles per gram wet weight) in liver of BALB/c Npc1 +/+ , Npc1 +/nih and Npc1 nih/nih male mice at 80 days of age (n = 3, mean ± SD). B: GlcChol (nanomoles per gram wet weight) in liver of C57BLKS Npc1 +/spm and Npc1 spm/spm male mice at 80 days of age (n = 3–5, mean ± SD). C: GlcChol (picomoles per milligram protein) in RAW264.7 cells incubated with the indicated concentration of U18666A for 1 day in the absence and presence of CBE-inhibiting GBA (n = 3, mean ± SD). D: GlcChol picomoles per milligram protein) in RAW264.7 cells incubated with 10 μM U18666A for 8 h and in the subsequent absence or presence of 1 mM β-methyl-cyclodextrin (β-mCD) reducing intralysosomal cholesterol for 18 h (n = 3, mean ± SD). Data were analyzed using an unpaired t -test. ** P
    Figure Legend Snippet: GlcChol abnormalities in NPC. A: GlcChol (nanomoles per gram wet weight) in liver of BALB/c Npc1 +/+ , Npc1 +/nih and Npc1 nih/nih male mice at 80 days of age (n = 3, mean ± SD). B: GlcChol (nanomoles per gram wet weight) in liver of C57BLKS Npc1 +/spm and Npc1 spm/spm male mice at 80 days of age (n = 3–5, mean ± SD). C: GlcChol (picomoles per milligram protein) in RAW264.7 cells incubated with the indicated concentration of U18666A for 1 day in the absence and presence of CBE-inhibiting GBA (n = 3, mean ± SD). D: GlcChol picomoles per milligram protein) in RAW264.7 cells incubated with 10 μM U18666A for 8 h and in the subsequent absence or presence of 1 mM β-methyl-cyclodextrin (β-mCD) reducing intralysosomal cholesterol for 18 h (n = 3, mean ± SD). Data were analyzed using an unpaired t -test. ** P

    Techniques Used: Mouse Assay, Incubation, Concentration Assay

    29) Product Images from "Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection"

    Article Title: Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.02694-15

    Alterations of cholesterol distribution and endosomal traffic with U18666A. Ctrl, cholesterol distribution in control cells with normal cholesterol efflux. CD63 and LBPA are stained in green and cholesterol in red. Upon U18666A treatment, inhibition of
    Figure Legend Snippet: Alterations of cholesterol distribution and endosomal traffic with U18666A. Ctrl, cholesterol distribution in control cells with normal cholesterol efflux. CD63 and LBPA are stained in green and cholesterol in red. Upon U18666A treatment, inhibition of

    Techniques Used: Staining, Inhibition

    A cholesterol efflux block impairs viral exit from endosomes. (A) Top panels, Rab7-labeled endosomes in control untreated, uninfected cells (DMSO), aggregates of dilated endosomes in uninfected cells treated with U18666A, and control infected, untreated
    Figure Legend Snippet: A cholesterol efflux block impairs viral exit from endosomes. (A) Top panels, Rab7-labeled endosomes in control untreated, uninfected cells (DMSO), aggregates of dilated endosomes in uninfected cells treated with U18666A, and control infected, untreated

    Techniques Used: Blocking Assay, Labeling, Infection

    30) Product Images from "Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation"

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709057

    Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.
    Figure Legend Snippet: Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.

    Techniques Used: Activation Assay, Incubation, Infection, Transfection

    Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P
    Figure Legend Snippet: Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P

    Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P
    Figure Legend Snippet: Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P

    Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Microscopy

    ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P
    Figure Legend Snippet: ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P

    Techniques Used: Labeling, Staining, Transfection

    31) Product Images from "The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus"

    Article Title: The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.01053-16

    U18666A treatment inhibits HCV particle secretion but not assembly in cell culture and primary cells. HCV-replicating cells were treated with 2 μg/ml of U18666A for 16 h. (A) The amount of released viral genomes in the supernatant was determined by RT-PCR. The graph shows the data from six independent experiments, with SEMs. (B) To determine the amount of released infectious viral particles in the supernatant, a TCID 50 assay was performed. The graph shows the relative data from three independent experiments, with SEMs. (C) The amount of released viral particles in the supernatant was determined by core Ag assay. The graph shows the data from seven independent experiments, with SEMs. (D) The amount of intracellular viral particles was assayed by TCID 50 of cells lysed by freeze-thawing. The graph shows the relative data from three independent experiments, with SEMs. (E) Primary human hepatocytes were infected over night with supernatant of HCV-replicating cells. Cells were treated 56 h p.i. with 2 μg/ml of U18666A for 16 h. Viral replication and particle release were determined by RT-PCR of cell lysates and supernatants, respectively. The graph shows the relative data from three independent experiments, with SEMs. (F) Huh7.5 cells were infected for 5 h with supernatant of HCV-replicating cells. To determine the entry-inhibiting ability of U18666A, it was added at a concentration of 2 μg/ml. As positive controls for entry inhibition, the supernatant was supplemented with 1 μg/ml of anti-E2 or anti-ApoE antibody. Bound virus was removed by short trypsin treatment before analysis of entered virus by RT-PCR. The graph shows the relative data from four independent experiments, with SEMs.
    Figure Legend Snippet: U18666A treatment inhibits HCV particle secretion but not assembly in cell culture and primary cells. HCV-replicating cells were treated with 2 μg/ml of U18666A for 16 h. (A) The amount of released viral genomes in the supernatant was determined by RT-PCR. The graph shows the data from six independent experiments, with SEMs. (B) To determine the amount of released infectious viral particles in the supernatant, a TCID 50 assay was performed. The graph shows the relative data from three independent experiments, with SEMs. (C) The amount of released viral particles in the supernatant was determined by core Ag assay. The graph shows the data from seven independent experiments, with SEMs. (D) The amount of intracellular viral particles was assayed by TCID 50 of cells lysed by freeze-thawing. The graph shows the relative data from three independent experiments, with SEMs. (E) Primary human hepatocytes were infected over night with supernatant of HCV-replicating cells. Cells were treated 56 h p.i. with 2 μg/ml of U18666A for 16 h. Viral replication and particle release were determined by RT-PCR of cell lysates and supernatants, respectively. The graph shows the relative data from three independent experiments, with SEMs. (F) Huh7.5 cells were infected for 5 h with supernatant of HCV-replicating cells. To determine the entry-inhibiting ability of U18666A, it was added at a concentration of 2 μg/ml. As positive controls for entry inhibition, the supernatant was supplemented with 1 μg/ml of anti-E2 or anti-ApoE antibody. Bound virus was removed by short trypsin treatment before analysis of entered virus by RT-PCR. The graph shows the relative data from four independent experiments, with SEMs.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Infection, Concentration Assay, Inhibition

    Preferentially, HCV particles with low density are retained intracellularly. (A) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The cells were lysed by repeated freeze-thaw cycles and loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs. (B) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The supernatant was loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs.
    Figure Legend Snippet: Preferentially, HCV particles with low density are retained intracellularly. (A) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The cells were lysed by repeated freeze-thaw cycles and loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs. (B) HCV J6-replicating Huh7.5 cells were treated with 2 μg/ml of U18666A for 16 h. The supernatant was loaded on an iodixanol gradient to separate viral particles by their density. Twelve fractions were harvested from top to bottom and were used to infect Huh7.5 cells. The infected cells were harvested 72 h p.i., and viral load was assessed by RT-PCR of the intracellular RNA. The top graph shows the relative infectivity of each fraction, and the bottom graph shows the infectivity as a percentage of the total measured infectivity. The graph shows the relative data from three independent experiments, with SEMs.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction

    LAMP2-positive rings are filled with lipids and could represent multilamellar bodies filled with HCV particles. (A) CLSM analysis of untreated HCV-replicating cells. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (B) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (C) CLSM analysis of HCV-replicating cells which were left untreated or treated with 2 μg/ml of U18666A for 16 h. ApoE is visualized in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. (D) TEM images of ultrathin sections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Ultrathin sections were stained with uranyl acetate to identify cellular and viral structures. (E) TEM image of cryosections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against LAMP2 or HERV-K as a negative control and stained with uranyl acetate. (F) TEM image of cryosections of Huh7.5 cells electroporated with J6 or GND which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against core and E2 and stained with uranyl acetate.
    Figure Legend Snippet: LAMP2-positive rings are filled with lipids and could represent multilamellar bodies filled with HCV particles. (A) CLSM analysis of untreated HCV-replicating cells. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (B) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. The lipid droplets were stained with BODIPY in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. The graph shows the fluorescence intensities of DAPI, BODIPY, and LAMP2 along the white arrow in the zoomed image. (C) CLSM analysis of HCV-replicating cells which were left untreated or treated with 2 μg/ml of U18666A for 16 h. ApoE is visualized in green, and the lysosomal marker LAMP2 is visualized in red. The nuclei were stained with DAPI in blue. (D) TEM images of ultrathin sections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Ultrathin sections were stained with uranyl acetate to identify cellular and viral structures. (E) TEM image of cryosections of HCV-replicating Huh7.5 cells which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against LAMP2 or HERV-K as a negative control and stained with uranyl acetate. (F) TEM image of cryosections of Huh7.5 cells electroporated with J6 or GND which were treated with 2 μg/ml of U18666A for 16 h. Sections were labeled with 10-nm-gold-conjugated antibodies against core and E2 and stained with uranyl acetate.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Marker, Fluorescence, Transmission Electron Microscopy, Labeling, Negative Control

    U18666A has no effect on NS5A localization, but ApoE accumulates and LAMP2 forms ring-like structures. Shown are results of CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. (A) NS5A is visualized in red; nuclei were stained with DAPI, in blue, and ApoE is visualized in green. (B) The fluorescence intensity of ApoE was measured in untreated and U18666A-treated cells. The data were confirmed by quantitative Western blot analysis of cellular lysates using ApoE- and actin-specific antibodies. The quantification is based on three independent experiments. (C) Pearson's overlap coefficient of NS5A and ApoE was calculated in untreated and U18666A-treated cells. (D) The amount of secreted ApoE was measured using quantitative Western blotting of TCA-precipitated samples of untreated and U18666A-treated cells. The quantification is based on three independent experiments. (E) NS5A is visualized in red; cholesterol was stained with filipin in cyan, and the lysosomal marker LAMP2 is visualized in green. The enlarged images are shown for treated and untreated cells. (F) The fluorescence intensity of LAMP2 was measured in untreated and U18666A-treated cells. The findings were confirmed by quantitative Western blotting of cell lysates. The quantification is based on three independent experiments. (G) Huh7.5 cells expressing Rab7-YFP were stained for LAMP2, in red. Treatment of U18666A led to the formation of Rab7- and LAMP2-positive rings.
    Figure Legend Snippet: U18666A has no effect on NS5A localization, but ApoE accumulates and LAMP2 forms ring-like structures. Shown are results of CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. (A) NS5A is visualized in red; nuclei were stained with DAPI, in blue, and ApoE is visualized in green. (B) The fluorescence intensity of ApoE was measured in untreated and U18666A-treated cells. The data were confirmed by quantitative Western blot analysis of cellular lysates using ApoE- and actin-specific antibodies. The quantification is based on three independent experiments. (C) Pearson's overlap coefficient of NS5A and ApoE was calculated in untreated and U18666A-treated cells. (D) The amount of secreted ApoE was measured using quantitative Western blotting of TCA-precipitated samples of untreated and U18666A-treated cells. The quantification is based on three independent experiments. (E) NS5A is visualized in red; cholesterol was stained with filipin in cyan, and the lysosomal marker LAMP2 is visualized in green. The enlarged images are shown for treated and untreated cells. (F) The fluorescence intensity of LAMP2 was measured in untreated and U18666A-treated cells. The findings were confirmed by quantitative Western blotting of cell lysates. The quantification is based on three independent experiments. (G) Huh7.5 cells expressing Rab7-YFP were stained for LAMP2, in red. Treatment of U18666A led to the formation of Rab7- and LAMP2-positive rings.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Fluorescence, Western Blot, Marker, Expressing

    Time- and dose-dependent inhibition of viral replication by U18666A. (A) Huh9-13 cells were treated with 10 μg/ml of simvastatin or the indicated concentrations of U18666A for 72 h. w/o, without treatment. Viral replication was determined by RT-PCR. The graph shows the relative data from three independent experiments, with SEMs. (B) Huh7.5 cells were transfected with the J6-Luc construct and treated with 1,000 U of interferon alpha, 10 μg/ml of simvastatin, or the indicated concentrations of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay. The graph shows the relative data from three independent experiments, with SEMs. (C) Huh7.5 cells were transfected with the J6-Luc construct and treated with 2 μg/ml of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay at different time points. The graph shows the relative data from three independent experiments, with SEMs. (D) Huh9-13 and Huh7.5 J6 cells were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. Viral replication was determined by RT-PCR. The graph shows the relative data from at least three independent experiments, with SEMs. ns, not significant. (E) Western blot analysis of cellular lysates derived from Huh9-13 and Huh7.5 J6 cells which were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. For detection, specific antibodies against NS3, NS5A, and core were used. Detection of β-actin served as a loading control. (F) Densitometric quantification of Western blots shown in panel E. The graphs show the quantification of at least three independent experiments, with SEMs.
    Figure Legend Snippet: Time- and dose-dependent inhibition of viral replication by U18666A. (A) Huh9-13 cells were treated with 10 μg/ml of simvastatin or the indicated concentrations of U18666A for 72 h. w/o, without treatment. Viral replication was determined by RT-PCR. The graph shows the relative data from three independent experiments, with SEMs. (B) Huh7.5 cells were transfected with the J6-Luc construct and treated with 1,000 U of interferon alpha, 10 μg/ml of simvastatin, or the indicated concentrations of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay. The graph shows the relative data from three independent experiments, with SEMs. (C) Huh7.5 cells were transfected with the J6-Luc construct and treated with 2 μg/ml of U18666A for 72 h. Viral replication was determined by luciferase reporter gene assay at different time points. The graph shows the relative data from three independent experiments, with SEMs. (D) Huh9-13 and Huh7.5 J6 cells were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. Viral replication was determined by RT-PCR. The graph shows the relative data from at least three independent experiments, with SEMs. ns, not significant. (E) Western blot analysis of cellular lysates derived from Huh9-13 and Huh7.5 J6 cells which were treated with 2 μg/ml of U18666A or 1,000 U of interferon alpha for 16 h. For detection, specific antibodies against NS3, NS5A, and core were used. Detection of β-actin served as a loading control. (F) Densitometric quantification of Western blots shown in panel E. The graphs show the quantification of at least three independent experiments, with SEMs.

    Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Luciferase, Reporter Gene Assay, Western Blot, Derivative Assay

    U18666A treatment leads to the formation of nonfunctional lysosomal MLBs in which the HCV particles accumulate. The viral RNA is transported from the replication complex (RC) to the assembly complex (AC) at the membranous web, where the particles are formed. VLDLs as well as LVPs reach the MVBs, where they are sorted for either release or degradation by fusion with lysosomes. Addition of U18666A disturbs this mechanism by blocking the secretion of cholesterol and exosomes by MVBs. This leads to an accumulation of these HCV particles determined for release in exosomes. As the endolysosomes are overloaded with cholesterol and other lipids, they form nonfunctional MLBs. The HCV particles that are determined for lysosomal degradation accumulate here and are prevented from degradation due to the dysfunctionality of the MLBs.
    Figure Legend Snippet: U18666A treatment leads to the formation of nonfunctional lysosomal MLBs in which the HCV particles accumulate. The viral RNA is transported from the replication complex (RC) to the assembly complex (AC) at the membranous web, where the particles are formed. VLDLs as well as LVPs reach the MVBs, where they are sorted for either release or degradation by fusion with lysosomes. Addition of U18666A disturbs this mechanism by blocking the secretion of cholesterol and exosomes by MVBs. This leads to an accumulation of these HCV particles determined for release in exosomes. As the endolysosomes are overloaded with cholesterol and other lipids, they form nonfunctional MLBs. The HCV particles that are determined for lysosomal degradation accumulate here and are prevented from degradation due to the dysfunctionality of the MLBs.

    Techniques Used: Blocking Assay

    HCV structural proteins accumulate intracellularly in endosomal structures. (A) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. HCV core is visualized in green; the nuclei were stained with DAPI, in blue, and the exosomal marker CD63 is visualized in red. Shown are the fluorescence intensities of DAPI, core, and CD63 along the white arrows. Enlarged images are shown for treated and untreated cells. (B) The fluorescence intensities of core and CD63 were measured in untreated and U18666A-treated cells (left). The amount of CD63 was also determined by quantitative Western blotting using cell lysates (right). The quantification is based on three independent experiments. (C) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml ofU18666A for 16 h. HCV core is visualized in red; the nuclei were stained with DAPI, in blue, and the autophagosomal marker LC3 is visualized in green. The graphs show the fluorescence intensities of DAPI, core, and LC3 along the white arrows in the zoomed image. The enlarged images are shown for treated and untreated cells. (D) The weighted colocalization coefficients of core and CD63 were calculated in untreated and U18666A-treated cells. (E) CLSM analysis of Huh7.5 cells that were electroporated with HCV J6 RNA and plasmids encoding the indicated fusion proteins. HCV core was costained with a specific antibody. (F) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. Huh7.5 cells were transfected with an HCV construct harboring an mCherry fusion tag inside the hypervariable region (HVR) of E1, which is visualized in red. The nuclei were stained with DAPI, in blue, and the lysosomal marker LAMP2 is visualized in green. (G) Zoomed image of a part of panel F with intensity profiles showing the fluorescence intensities of DAPI, LAMP2, and E1 along the white arrows.
    Figure Legend Snippet: HCV structural proteins accumulate intracellularly in endosomal structures. (A) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. HCV core is visualized in green; the nuclei were stained with DAPI, in blue, and the exosomal marker CD63 is visualized in red. Shown are the fluorescence intensities of DAPI, core, and CD63 along the white arrows. Enlarged images are shown for treated and untreated cells. (B) The fluorescence intensities of core and CD63 were measured in untreated and U18666A-treated cells (left). The amount of CD63 was also determined by quantitative Western blotting using cell lysates (right). The quantification is based on three independent experiments. (C) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml ofU18666A for 16 h. HCV core is visualized in red; the nuclei were stained with DAPI, in blue, and the autophagosomal marker LC3 is visualized in green. The graphs show the fluorescence intensities of DAPI, core, and LC3 along the white arrows in the zoomed image. The enlarged images are shown for treated and untreated cells. (D) The weighted colocalization coefficients of core and CD63 were calculated in untreated and U18666A-treated cells. (E) CLSM analysis of Huh7.5 cells that were electroporated with HCV J6 RNA and plasmids encoding the indicated fusion proteins. HCV core was costained with a specific antibody. (F) CLSM analysis of HCV-replicating cells which were treated with 2 μg/ml of U18666A for 16 h. Huh7.5 cells were transfected with an HCV construct harboring an mCherry fusion tag inside the hypervariable region (HVR) of E1, which is visualized in red. The nuclei were stained with DAPI, in blue, and the lysosomal marker LAMP2 is visualized in green. (G) Zoomed image of a part of panel F with intensity profiles showing the fluorescence intensities of DAPI, LAMP2, and E1 along the white arrows.

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Marker, Fluorescence, Western Blot, Transfection, Construct

    32) Product Images from "Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes *"

    Article Title: Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.014993

    Treatment with the amino steroid U18666A of HeLa cells stably expressing SLP-1-GFP results in the formation of large, cholesterol-rich vesicles. A , upper panel , a mixture of control and SLP-1-GFP expressing HeLa cells was cultivated in the absence of
    Figure Legend Snippet: Treatment with the amino steroid U18666A of HeLa cells stably expressing SLP-1-GFP results in the formation of large, cholesterol-rich vesicles. A , upper panel , a mixture of control and SLP-1-GFP expressing HeLa cells was cultivated in the absence of

    Techniques Used: Stable Transfection, Expressing

    Reduced LAMP-2 expression on the large, cholesterol-rich SLP-1-GFP-positive vesicles. Cells stably expressing either SLP-1-GFP or SLP-1-(1–288)-GFP were treated with 3 μg/ml U18666A for 24 h. The cells were fixed and stained with filipin
    Figure Legend Snippet: Reduced LAMP-2 expression on the large, cholesterol-rich SLP-1-GFP-positive vesicles. Cells stably expressing either SLP-1-GFP or SLP-1-(1–288)-GFP were treated with 3 μg/ml U18666A for 24 h. The cells were fixed and stained with filipin

    Techniques Used: Expressing, Stable Transfection, Staining

    33) Product Images from "Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation"

    Article Title: Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2010.10.030

    Augmented lysosomal cholesterol content results in increased lysosomal stability. Normal human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) before exposure to MSDH (15 μmol/L). A: Immunocytochemical staining of cathepsin
    Figure Legend Snippet: Augmented lysosomal cholesterol content results in increased lysosomal stability. Normal human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) before exposure to MSDH (15 μmol/L). A: Immunocytochemical staining of cathepsin

    Techniques Used: Staining

    U18666A treatment is not associated with altered expression or localization of Bcl-2, Bcl-X L , Mcl-1, or Hsp70 but increased LC3-II levels. Fibroblasts were pretreated with 0.5 μg/ml U18666A for the indicated times. A: Expression of antiapoptotic
    Figure Legend Snippet: U18666A treatment is not associated with altered expression or localization of Bcl-2, Bcl-X L , Mcl-1, or Hsp70 but increased LC3-II levels. Fibroblasts were pretreated with 0.5 μg/ml U18666A for the indicated times. A: Expression of antiapoptotic

    Techniques Used: Expressing

    Fibroblasts with high cholesterol content are protected from apoptosis induced by MSDH. Human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) to induce lysosomal cholesterol accumulation before exposure to the lysosomotropic detergent
    Figure Legend Snippet: Fibroblasts with high cholesterol content are protected from apoptosis induced by MSDH. Human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) to induce lysosomal cholesterol accumulation before exposure to the lysosomotropic detergent

    Techniques Used:

    Fibroblasts with high cholesterol content are less sensitive to apoptosis induced by STS and cisplatin. Human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) to induce lysosomal cholesterol accumulation before exposure to STS (0.1
    Figure Legend Snippet: Fibroblasts with high cholesterol content are less sensitive to apoptosis induced by STS and cisplatin. Human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) to induce lysosomal cholesterol accumulation before exposure to STS (0.1

    Techniques Used:

    U18666A treatment results in cholesterol accumulation and lysosomal alterations but does not affect cell viability. Human fibroblasts were treated with 0.5 μg/ml U18666A for the indicated times. A: Intracellular cholesterol content of cells exposed
    Figure Legend Snippet: U18666A treatment results in cholesterol accumulation and lysosomal alterations but does not affect cell viability. Human fibroblasts were treated with 0.5 μg/ml U18666A for the indicated times. A: Intracellular cholesterol content of cells exposed

    Techniques Used:

    34) Product Images from "Niemann-Pick type C2 deficiency impairs autophagy-lysosomal activity, mitochondrial function, and TLR signaling in adipocytes"

    Article Title: Niemann-Pick type C2 deficiency impairs autophagy-lysosomal activity, mitochondrial function, and TLR signaling in adipocytes

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M066522

    NPC2 kd in 3T3-L1 adipocytes. A: NPC2 protein expression in different tissues. B: The gene expression of Npc2 in 3T3-L1 cells during differentiation. C: Npc2 mRNA expression in 3T3-L1 adipocytes was quantified by quantitative PCR. D: Protein expression of NPC2 in 3T3-L1 adipocytes by Western blot. E: Oil Red O staining of 3T3-L1 cells on day 8 of differentiation. F: Adipogenic gene expression in 3T3-L1 adipocytes on day 8 of differentiation (n = 4 in each group). G: Filipin III fluorescence image of scrambled and NPC2-kd adipocytes treated with 10 μg/ml U18666a for 48 and 72 h, respectively. Images were acquired using the automatic fluorescence microscope at 20× magnification. Scale bar = 50 μm. Data are expressed relative to the value for scrambled (Scr) cells. The values are mean ± SEM. * P
    Figure Legend Snippet: NPC2 kd in 3T3-L1 adipocytes. A: NPC2 protein expression in different tissues. B: The gene expression of Npc2 in 3T3-L1 cells during differentiation. C: Npc2 mRNA expression in 3T3-L1 adipocytes was quantified by quantitative PCR. D: Protein expression of NPC2 in 3T3-L1 adipocytes by Western blot. E: Oil Red O staining of 3T3-L1 cells on day 8 of differentiation. F: Adipogenic gene expression in 3T3-L1 adipocytes on day 8 of differentiation (n = 4 in each group). G: Filipin III fluorescence image of scrambled and NPC2-kd adipocytes treated with 10 μg/ml U18666a for 48 and 72 h, respectively. Images were acquired using the automatic fluorescence microscope at 20× magnification. Scale bar = 50 μm. Data are expressed relative to the value for scrambled (Scr) cells. The values are mean ± SEM. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Fluorescence, Microscopy

    35) Product Images from "Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway"

    Article Title: Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

    Journal: mBio

    doi: 10.1128/mBio.01857-15

    Lipid mixing requires EBOV GP-NPC1 interaction. (A) Lipid mixing by EBOV GPΔMuc-pseudotyped virus is significantly reduced in U2OS cells lacking the NPC1 cholesterol transporter. Asterisks indicate that values are significantly lower than those seen with wt U2OS. (B) A GP mutant unable to bind NPC1 displays impaired lipid mixing activity and infectivity. Asterisks indicate that values are significantly lower than those seen with VSV-EBOV GPΔMuc. (C) Lipid mixing is decreased by addition of the drug U18666A (10 µM), which inhibits cholesterol transport from within endosomes and induces an NPC1 disease phenotype. Asterisks indicate that values are significantly lower than those seen with the untreated control.
    Figure Legend Snippet: Lipid mixing requires EBOV GP-NPC1 interaction. (A) Lipid mixing by EBOV GPΔMuc-pseudotyped virus is significantly reduced in U2OS cells lacking the NPC1 cholesterol transporter. Asterisks indicate that values are significantly lower than those seen with wt U2OS. (B) A GP mutant unable to bind NPC1 displays impaired lipid mixing activity and infectivity. Asterisks indicate that values are significantly lower than those seen with VSV-EBOV GPΔMuc. (C) Lipid mixing is decreased by addition of the drug U18666A (10 µM), which inhibits cholesterol transport from within endosomes and induces an NPC1 disease phenotype. Asterisks indicate that values are significantly lower than those seen with the untreated control.

    Techniques Used: Mutagenesis, Activity Assay, Infection

    36) Product Images from "Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway ▿"

    Article Title: Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01062-08

    CD59 delivery to the inclusion does not involve the Golgi apparatus or multivesicular bodies. (A) HeLa cells grown on coverslips were treated at 1 hpi with brefeldin A or nocodazole to disrupt the organization and function of the Golgi apparatus or with LY294002 and U18666A to prevent multivesicular body formation. Cells were stained with antibodies to CD59 and IncA. Arrows indicate inclusions that stained for both CD59 and IncA. The effectiveness of brefeldin A and nocodazole treatments was confirmed on parallel coverslips by visualizing disruption of normal Golgi apparatus organization with antibodies to the Golgi marker protein Golgin97. Bar, 10 μm. (B) Percentages of IncA-positive cells in which CD59 was detected at the inclusion after treatment with the above pharmacologic inhibitors. An average of 50 cells were counted for each of the three independent experiments, and the mean and standard deviation were calculated. Fisher's exact test was used to determine P values for brefeldin A (0.2008), nocodazole (0.2210), LY294002 (0.0002), and U18666A (0.0164).
    Figure Legend Snippet: CD59 delivery to the inclusion does not involve the Golgi apparatus or multivesicular bodies. (A) HeLa cells grown on coverslips were treated at 1 hpi with brefeldin A or nocodazole to disrupt the organization and function of the Golgi apparatus or with LY294002 and U18666A to prevent multivesicular body formation. Cells were stained with antibodies to CD59 and IncA. Arrows indicate inclusions that stained for both CD59 and IncA. The effectiveness of brefeldin A and nocodazole treatments was confirmed on parallel coverslips by visualizing disruption of normal Golgi apparatus organization with antibodies to the Golgi marker protein Golgin97. Bar, 10 μm. (B) Percentages of IncA-positive cells in which CD59 was detected at the inclusion after treatment with the above pharmacologic inhibitors. An average of 50 cells were counted for each of the three independent experiments, and the mean and standard deviation were calculated. Fisher's exact test was used to determine P values for brefeldin A (0.2008), nocodazole (0.2210), LY294002 (0.0002), and U18666A (0.0164).

    Techniques Used: Staining, Marker, Standard Deviation

    37) Product Images from "The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages"

    Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029042

    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 Legend Snippet: 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.

    Techniques Used: Incubation, Staining, Epifluorescence Microscopy

    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.
    Figure Legend Snippet: 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.

    Techniques Used: Incubation, Expressing

    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 Legend Snippet: 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.

    Techniques Used: Activity Assay, Incubation

    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 Legend Snippet: 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.

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    38) Product Images from "Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation"

    Article Title: Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17071122

    Overexpression of NPC2 diminishes U18666A- and TGF-β1-induced free cholesterol accumulation and HSCs activation. ( A , B ) Different lentiviruses infected stable cells were pretreated with or without 1 µM U18666A overnight and then subjected to intracellular free cholesterol quantification; ( C – E ) eGFP- and NPC2-overexpressed LX2 cells were pretreated with 1 µM U18666A overnight and then treated with 20 ng/mL TGF-β1. Cell lysates were immunoblotted to detect α-SMA ( C ) and Smad2 phosphorylation (►, Smad2, ►►, Smad3) ( E ). mRNA expression of Col1a2 was analyzed using Q-PCR ( D ). * p
    Figure Legend Snippet: Overexpression of NPC2 diminishes U18666A- and TGF-β1-induced free cholesterol accumulation and HSCs activation. ( A , B ) Different lentiviruses infected stable cells were pretreated with or without 1 µM U18666A overnight and then subjected to intracellular free cholesterol quantification; ( C – E ) eGFP- and NPC2-overexpressed LX2 cells were pretreated with 1 µM U18666A overnight and then treated with 20 ng/mL TGF-β1. Cell lysates were immunoblotted to detect α-SMA ( C ) and Smad2 phosphorylation (►, Smad2, ►►, Smad3) ( E ). mRNA expression of Col1a2 was analyzed using Q-PCR ( D ). * p

    Techniques Used: Over Expression, Activation Assay, Infection, Expressing, Polymerase Chain Reaction

    39) Product Images from "Novel mechanism of U18666A-induced tumour necrosis factor-α production in RAW 264·7 macrophage cells"

    Article Title: Novel mechanism of U18666A-induced tumour necrosis factor-α production in RAW 264·7 macrophage cells

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2008.03779.x

    Tumour necrosis factor (TNF)-α production in U18666A-treated RAW 264·7 cells in medium X and Y. RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the presence or absence of N-acetyl-L-cysteine (5 mM) in medium X and Y. * P
    Figure Legend Snippet: Tumour necrosis factor (TNF)-α production in U18666A-treated RAW 264·7 cells in medium X and Y. RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the presence or absence of N-acetyl-L-cysteine (5 mM) in medium X and Y. * P

    Techniques Used: Incubation

    Participation of p38 in U18666A-induced tumour necrosis factor (TNF)-α production. RAW 264·7 cells were incubated with U18666A (1 µg/ml) in the presence of SB203580 (10 µM) or PD98058 (10 µM). In one of the experimental groups SB203580 was added into the culture 12 h after U18666A treatment. TNF-α production was determined with enzyme-linked immunosorbent assay. * P
    Figure Legend Snippet: Participation of p38 in U18666A-induced tumour necrosis factor (TNF)-α production. RAW 264·7 cells were incubated with U18666A (1 µg/ml) in the presence of SB203580 (10 µM) or PD98058 (10 µM). In one of the experimental groups SB203580 was added into the culture 12 h after U18666A treatment. TNF-α production was determined with enzyme-linked immunosorbent assay. * P

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    Oxidative stress-responsive signal transduction in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours. (b) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the presence of N-acetyl-L-cysteine (5 mM) or medium Y. The phosphorylation was detected by immunoblotting.
    Figure Legend Snippet: Oxidative stress-responsive signal transduction in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours. (b) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the presence of N-acetyl-L-cysteine (5 mM) or medium Y. The phosphorylation was detected by immunoblotting.

    Techniques Used: Transduction, Incubation

    Reactive oxygen species generation in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the culture with medium X or medium Y. * P
    Figure Legend Snippet: Reactive oxygen species generation in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the culture with medium X or medium Y. * P

    Techniques Used: Incubation

    Tumour necrosis factor (TNF)-α production in U18666A-treated RAW 264·7 cells. RAW 264·7 cells and peritoneal macrophages were incubated with U18666A (1 µg/ml) for various hours. (a) TNF-α production was determined with enzyme-linked immunosorbent assay. * P
    Figure Legend Snippet: Tumour necrosis factor (TNF)-α production in U18666A-treated RAW 264·7 cells. RAW 264·7 cells and peritoneal macrophages were incubated with U18666A (1 µg/ml) for various hours. (a) TNF-α production was determined with enzyme-linked immunosorbent assay. * P

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    Intracellular free cholesterol accumulation in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the culture with medium X or medium Y. * P
    Figure Legend Snippet: Intracellular free cholesterol accumulation in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the culture with medium X or medium Y. * P

    Techniques Used: Incubation

    40) Product Images from "Adenovirus RID-? activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C"

    Article Title: Adenovirus RID-? activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200903039

    RID-α (C67S) induces the formation of enlarged lipid-filled LAMP1 structures. (a) Confocal images of CHO cell lines stained with LAMP1 antibody and filipin. (b and c) Magnified images of single and merged channels from CHO–RID-α (C67S) cells stained with LBPA antibody and filipin (b) or LBPA antibody after incubation with Alexa Fluor 647 CT-B (c). (d) CHO cell lines treated with U18666A for 8 h and stained with LAMP1 antibody and filipin. (e) Cholesterol quantification in CHO cell lines treated with DMSO (vehicle) or U18666A for 8 h using the Amplex red cholesterol assay kit. Values were normalized to total cellular protein and are displayed as mean ± SEM (*, P
    Figure Legend Snippet: RID-α (C67S) induces the formation of enlarged lipid-filled LAMP1 structures. (a) Confocal images of CHO cell lines stained with LAMP1 antibody and filipin. (b and c) Magnified images of single and merged channels from CHO–RID-α (C67S) cells stained with LBPA antibody and filipin (b) or LBPA antibody after incubation with Alexa Fluor 647 CT-B (c). (d) CHO cell lines treated with U18666A for 8 h and stained with LAMP1 antibody and filipin. (e) Cholesterol quantification in CHO cell lines treated with DMSO (vehicle) or U18666A for 8 h using the Amplex red cholesterol assay kit. Values were normalized to total cellular protein and are displayed as mean ± SEM (*, P

    Techniques Used: Staining, Incubation, Amplex Red Cholesterol Assay

    Related Articles

    Transfection:

    Article Title: ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease
    Article Snippet: .. After 2 h, transfection complexes were removed, and cells were incubated with fresh complete DMEM containing 3 H-cholesterol (1 µCi/ml), 0.2% BSA and U18666A for 4–6 hours. ..

    In Vitro:

    Article Title: Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease
    Article Snippet: .. In vitro the NPC phenotype was induced with U18666A (Sigma) at concentrations ranging from 1–10 μM; N-butyl-1-deoxynojirimycin (Sigma) was used at 250 μM to block GSL synthesis. .. Animals Npc1 nih/nih and mice Npc1 nmf164 , along with wild-type littermates (Npc1 +/+ ), were generated by crossing Npc1 +/nih or Npc1 +/nmf164 males and females in-house.

    Infection:

    Article Title: The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux
    Article Snippet: .. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection . ..

    Incubation:

    Article Title: ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease
    Article Snippet: .. After 2 h, transfection complexes were removed, and cells were incubated with fresh complete DMEM containing 3 H-cholesterol (1 µCi/ml), 0.2% BSA and U18666A for 4–6 hours. ..

    other:

    Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
    Article Snippet: Dimethylsufoxide (DMSO) was from ATCC (Manassas, VA); 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) was from Sigma (St. Louis, MO); LY294002, U18666A, bafilomycin A, dynasore, nystatin, and chlorpromazine hydrochloride (CPZ) were from EMD Millipore (Billerica, MA).

    Blocking Assay:

    Article Title: Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease
    Article Snippet: .. In vitro the NPC phenotype was induced with U18666A (Sigma) at concentrations ranging from 1–10 μM; N-butyl-1-deoxynojirimycin (Sigma) was used at 250 μM to block GSL synthesis. .. Animals Npc1 nih/nih and mice Npc1 nmf164 , along with wild-type littermates (Npc1 +/+ ), were generated by crossing Npc1 +/nih or Npc1 +/nmf164 males and females in-house.

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation
    Article Snippet: .. Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation. .. Primary BMDMs exposed to U18666a displayed lysosomal cholesterol accumulation at 48 h, while in iBMDMs, sufficient cholesterol accumulation was observed by 24 h (Fig. S1).

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    Millipore u18666a
    Increased GPMV formation preceeds efflux of cholesterol in an NPC1 model. A: GM03123 were loaded with TopFluor-cholesterol and treated as indicated. Measurement of fluorescence in media shows an increase in cholesterol efflux elicited by MβCD treatment and inhibition by <t>U18666A.</t> B: Quantification of intracellular cholesterol in GM03123 fibroblasts treated with MβCD for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. C: Quantification of intracellular cholesterol in GM03123 treated with LXR agonist GW3965 for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. D: Cells treated with GW3965 for 24 hours show an increase in GPMV formation. Cells stained with filipin. Arrows indicate GPMVs. All error bars indicate the standard error of the mean.
    U18666a, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Increased GPMV formation preceeds efflux of cholesterol in an NPC1 model. A: GM03123 were loaded with TopFluor-cholesterol and treated as indicated. Measurement of fluorescence in media shows an increase in cholesterol efflux elicited by MβCD treatment and inhibition by U18666A. B: Quantification of intracellular cholesterol in GM03123 fibroblasts treated with MβCD for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. C: Quantification of intracellular cholesterol in GM03123 treated with LXR agonist GW3965 for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. D: Cells treated with GW3965 for 24 hours show an increase in GPMV formation. Cells stained with filipin. Arrows indicate GPMVs. All error bars indicate the standard error of the mean.

    Journal: Experimental cell research

    Article Title: The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

    doi: 10.1016/j.yexcr.2018.03.001

    Figure Lengend Snippet: Increased GPMV formation preceeds efflux of cholesterol in an NPC1 model. A: GM03123 were loaded with TopFluor-cholesterol and treated as indicated. Measurement of fluorescence in media shows an increase in cholesterol efflux elicited by MβCD treatment and inhibition by U18666A. B: Quantification of intracellular cholesterol in GM03123 fibroblasts treated with MβCD for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. C: Quantification of intracellular cholesterol in GM03123 treated with LXR agonist GW3965 for 72 hours. Corrected total cell fluorescence of filipin shows a decrease in cellular cholesterol levels. D: Cells treated with GW3965 for 24 hours show an increase in GPMV formation. Cells stained with filipin. Arrows indicate GPMVs. All error bars indicate the standard error of the mean.

    Article Snippet: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection .

    Techniques: Fluorescence, Inhibition, Staining

    GPMV formation as a visible readout of cholesterol efflux. A: Treatment of cells with U18666A to aggregate cholesterol intracellularly inhibits GPMV formation. Treatment with water-soluble cholesterol (cholesterol-MβCD) to augment the cholesterol pool increases GPMV formation. Cells labeled with filipin. GPMVs indicated by arrows. B: ) and the corrected total cell fluorescence for cells treated with ApoA1 at 48 hours is shown. Error bars indicate the standard error of the mean. C. HeLa treated with ApoA1 for 24 and 48 hours show increased GPMV formation (arrows) most prominently at 24 hours, and decreased cellular cholesterol levels at 48 hours. Cells labeled with filipin.

    Journal: Experimental cell research

    Article Title: The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

    doi: 10.1016/j.yexcr.2018.03.001

    Figure Lengend Snippet: GPMV formation as a visible readout of cholesterol efflux. A: Treatment of cells with U18666A to aggregate cholesterol intracellularly inhibits GPMV formation. Treatment with water-soluble cholesterol (cholesterol-MβCD) to augment the cholesterol pool increases GPMV formation. Cells labeled with filipin. GPMVs indicated by arrows. B: ) and the corrected total cell fluorescence for cells treated with ApoA1 at 48 hours is shown. Error bars indicate the standard error of the mean. C. HeLa treated with ApoA1 for 24 and 48 hours show increased GPMV formation (arrows) most prominently at 24 hours, and decreased cellular cholesterol levels at 48 hours. Cells labeled with filipin.

    Article Snippet: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection .

    Techniques: Labeling, Fluorescence

    Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.

    Journal: The Journal of Cell Biology

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    doi: 10.1083/jcb.201709057

    Figure Lengend Snippet: Npc1 deficiency does not affect the activation of NLRC4 and AIM2 inflammasomes. (A) WT iBMDMs were incubated with or without the presence of increasing concentrations of U18666a (2, 5, and 10 µg/ml) alongside Nlrc4 −/− cells and subsequently infected with S. typhimurium at an MOI of 2 for ∼4 h. Cell lysates were immunoblotted for casp-1 and GAPDH. (B) WT and Npc1 −/− cells were treated with S. typhimurium for 4 h and immunoblotted as in A. (C) Cell supernatants were analyzed for IL-1β. (D) WT cells either treated or not with U18666a (5 µg/ml) and Npc1 −/− cells were transfected with poly(dA:dT) for 4 h before cell lysates were immunoblotted for the antibodies indicated. (E) WT, Asc −/− , and caspase 1/11 −/− cells were exposed or not to U18666a before infection with S. typhimurium as above. Cell lysates were immunoblotted for GSDMD and GAPDH. (F and G) BMDMs were treated with either LPS (500 ng/ml; 4 h) or Pam3 (500 ng/ml; 4 h) in the presence of increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) followed by ATP (5 mM; 45 min). Cell lysates were immunoblotted for GSDMD and GAPDH.

    Article Snippet: Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation.

    Techniques: Activation Assay, Incubation, Infection, Transfection

    Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P

    Journal: The Journal of Cell Biology

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    doi: 10.1083/jcb.201709057

    Figure Lengend Snippet: Cholesterol supplementation restores inflammasome activation in cells defective in NPC1 function. (A) BMDMs were either left untreated or exposed to LPS and cholesterol–MCD complexes (chol), cholesterol and ATP, or LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with 15 µg/ml cholesterol for 1 h before ATP treatment. Samples were immunoblotted with casp-1, and GAPDH was used as a loading control. (B) Cell supernatants were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. (C) IL-1β levels in LPS-primed BMDMs grown in complete DMEM and exposed to alum (1 mg/ml). (D) IL-1β levels in Npc1 −/− cells either left untreated or treated with LPS and cholesterol–MCD for 1 h followed by ATP. (E) BMDMs were either left untreated or exposed to LPS and ATP in the presence or absence of 5 µg/ml U18666a. Where added, cells were incubated with indicated concentrations of cholesterol–MCD for 1 h before ATP treatment. Samples were immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (F) Cell supernatants from above were analyzed for secreted IL-1β by ELISA. Bar graph shows percent IL-1β restoration when cholesterol–MCD was added. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. **, P

    Article Snippet: Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation.

    Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    doi: 10.1083/jcb.201709057

    Figure Lengend Snippet: Lysosomal sterol accumulation dampens inflammasome activation. (A) BMDMs were incubated with U18666a in the presence or absence of LPS followed by measurement of total cholesterol. (B) BMDMs were either left untreated or exposed to 5 µg/ml U18666a before treating them with LPS (500 ng/ml; 4 h) and ATP (5 mM; 45 min). Cell lysates were immunoblotted for casp-1 antibody, and GAPDH was used as loading control. (C) IL-1β release from cells treated as above. (D) BMDMs were either left untreated or exposed to increasing concentrations of U18666a (1, 2, 5, and 10 µg/ml) before stimulating them with LPS and ATP. (E and F) Cell lysates were immunoblotted for the antibodies indicated, and cell supernatants were analyzed for IL-1β (E) or IL-18 (F) by ELISA. (G) Microscopy images of cells treated as above in B or with Pam3 (500 ng/ml; 4 h) followed by ATP. Arrows show characteristic pyroptotic cell death. Bars, 20 µm. (H and I) LDH release in supernatants from cells treated as in G. (J and K) LPS-primed BMDMs treated with indicated concentrations of Baf A1 (J) or CQ (K) followed by ATP. Data shown are mean ± SD, and experiments shown are representative of at least three independent experiments. *, P

    Article Snippet: Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation.

    Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Microscopy

    ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P

    Journal: The Journal of Cell Biology

    Article Title: Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

    doi: 10.1083/jcb.201709057

    Figure Lengend Snippet: ER cholesterol depletion blunts ASC-dependent inflammasome assembly. (A) WT (control), U18666a-treated, and Npc1 −/− cells were exposed to LPS + ATP followed by labeling with anti-ASC antibody and DAPI staining. (B) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 40 cells. (C and D) LPS-primed BMDMs exposed to ATP (C) or poly(dA:dT)-transfected BMDMs (D) were exposed or not to lovastatin (40 µM; 1 h) followed by labeling with anti-ASC antibody and DAPI staining. (E) Quantitative analysis of percentage of cells with ASC specks in samples treated as above. Each dot represents an individual field with at least n = 30 cells. Data shown are mean ± SEM, and experiments shown are representative of at least three independent experiments. Arrowheads show ASC specks. Bars, 5 µm. ****, P

    Article Snippet: Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation.

    Techniques: Labeling, Staining, Transfection

    Lipid transport out of MVBs is dispensable for CCHFV entry. (A) SW13 cells were pretreated with U18666A (30 µM) for 1 h or left untreated (mock). Then, the cells were incubated with CCHFV in the presence of the drug for 24 h and subsequently fixed, permeabilized, and stained with anti-N antibody (red), anti-CD63 antibody (green), and CellMask blue dye (grey) to define cell boundaries. The samples were imaged by immunofluorescence, and an optical section through the middle of the cell is shown (left and middle panels). Relative infection efficiencies were calculated by dividing the number of infected cells by the total number of cells and are averages of three independent experiments, with error bars representing standard deviations (right panel). (B) Cells treated as described in (A) were fixed 1 h after treatment and then stained with anti-CD63 antibody (green), filipin III (red), and CellMask red dye (grey). The images were generated as described above. (C) SW13 cells were treated with U18666A (30 µM) for 1 h or left untreated (mock), then incubated with VSV-CCHFVG, VSV-EBOVGP, or VSV-LASVGP. Luciferase activity was measured 24 h after pseudotype addition.

    Journal: PLoS Pathogens

    Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators

    doi: 10.1371/journal.ppat.1004390

    Figure Lengend Snippet: Lipid transport out of MVBs is dispensable for CCHFV entry. (A) SW13 cells were pretreated with U18666A (30 µM) for 1 h or left untreated (mock). Then, the cells were incubated with CCHFV in the presence of the drug for 24 h and subsequently fixed, permeabilized, and stained with anti-N antibody (red), anti-CD63 antibody (green), and CellMask blue dye (grey) to define cell boundaries. The samples were imaged by immunofluorescence, and an optical section through the middle of the cell is shown (left and middle panels). Relative infection efficiencies were calculated by dividing the number of infected cells by the total number of cells and are averages of three independent experiments, with error bars representing standard deviations (right panel). (B) Cells treated as described in (A) were fixed 1 h after treatment and then stained with anti-CD63 antibody (green), filipin III (red), and CellMask red dye (grey). The images were generated as described above. (C) SW13 cells were treated with U18666A (30 µM) for 1 h or left untreated (mock), then incubated with VSV-CCHFVG, VSV-EBOVGP, or VSV-LASVGP. Luciferase activity was measured 24 h after pseudotype addition.

    Article Snippet: Dimethylsufoxide (DMSO) was from ATCC (Manassas, VA); 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) was from Sigma (St. Louis, MO); LY294002, U18666A, bafilomycin A, dynasore, nystatin, and chlorpromazine hydrochloride (CPZ) were from EMD Millipore (Billerica, MA).

    Techniques: Incubation, Staining, Immunofluorescence, Infection, Generated, Luciferase, Activity Assay

    Constitutively active ARF6 increases cholesterol removal in NPC-like cells. A , HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B , HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C , Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods ). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).

    Journal: PLoS ONE

    Article Title: ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease

    doi: 10.1371/journal.pone.0005193

    Figure Lengend Snippet: Constitutively active ARF6 increases cholesterol removal in NPC-like cells. A , HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B , HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C , Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods ). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).

    Article Snippet: After 2 h, transfection complexes were removed, and cells were incubated with fresh complete DMEM containing 3 H-cholesterol (1 µCi/ml), 0.2% BSA and U18666A for 4–6 hours.

    Techniques: Staining, Transfection, Mutagenesis, Expressing, Quantitation Assay, Two Tailed Test