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 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u18666a - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    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 "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

    12) 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

    13) 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

    14) 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

    15) 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

    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 "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

    18) 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

    19) 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

    20) 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:

    21) 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

    22) 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

    23) 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

    24) 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

    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 "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

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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:

    35) 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

    36) Product Images from "Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis"

    Article Title: Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis

    Journal: Journal of Virology

    doi: 10.1128/JVI.01196-17

    Pharmacological inhibition of NPC1 and its effects on HCV RNA replication and virus production. (A) The experimental setup is shown in the upper left panel. Huh7/Lunet cells were electroporated with the Renilla luciferase (RLuc) reporter virus JcR2A and 24 h later treated with U18666A, Ro 48-8071, or water (as a control), using the concentrations specified at the bottom of the bar graphs, for 24, 48, or 96 h. Cells were harvested for luciferase assay, whereas supernatants (SN) were transferred onto naive Huh7.5 cells that were harvested 48 h later for luciferase assay, thus allowing determinations of viral replication and virus production, respectively. The impact of drug treatment on cell viability was assessed by quantifying the intracellular ATP content. Means and standard deviations for 3 or 4 independent experiments are shown. (B and C) Effect of U18666A or Ro-48 8071 treatment on persistent HCV replication. Huh7 cells containing a stable luciferase-encoding subgenomic replicon of genotype 2a (LucUbiNeo_JFH1) or genotype 1b (LucUbiNeo_Con1ET) were treated for 48 h with the given drug concentrations. RNA replication was assessed by measuring firefly luciferase activity in cell lysates. Means and standard deviations for 4 or 5 experiments are depicted. (D and E) Genotype-dependent effect of U18666A or Ro 48-8071 treatment on de novo HCV RNA replication. Huh7/Lunet cells were transfected with subgenomic firefly luciferase reporter replicons of genotypes 1a, 1b, 2a, and 3a, and 4 h later, drugs were added to the medium at the concentrations specified at the bottom of the graphs. HCV replication was determined by luciferase assay 48 h after transfection. Cell viability in the same lysate was assessed as described above. Means and standard deviations for 4 independent experiments are shown. RLU, relative light units.
    Figure Legend Snippet: Pharmacological inhibition of NPC1 and its effects on HCV RNA replication and virus production. (A) The experimental setup is shown in the upper left panel. Huh7/Lunet cells were electroporated with the Renilla luciferase (RLuc) reporter virus JcR2A and 24 h later treated with U18666A, Ro 48-8071, or water (as a control), using the concentrations specified at the bottom of the bar graphs, for 24, 48, or 96 h. Cells were harvested for luciferase assay, whereas supernatants (SN) were transferred onto naive Huh7.5 cells that were harvested 48 h later for luciferase assay, thus allowing determinations of viral replication and virus production, respectively. The impact of drug treatment on cell viability was assessed by quantifying the intracellular ATP content. Means and standard deviations for 3 or 4 independent experiments are shown. (B and C) Effect of U18666A or Ro-48 8071 treatment on persistent HCV replication. Huh7 cells containing a stable luciferase-encoding subgenomic replicon of genotype 2a (LucUbiNeo_JFH1) or genotype 1b (LucUbiNeo_Con1ET) were treated for 48 h with the given drug concentrations. RNA replication was assessed by measuring firefly luciferase activity in cell lysates. Means and standard deviations for 4 or 5 experiments are depicted. (D and E) Genotype-dependent effect of U18666A or Ro 48-8071 treatment on de novo HCV RNA replication. Huh7/Lunet cells were transfected with subgenomic firefly luciferase reporter replicons of genotypes 1a, 1b, 2a, and 3a, and 4 h later, drugs were added to the medium at the concentrations specified at the bottom of the graphs. HCV replication was determined by luciferase assay 48 h after transfection. Cell viability in the same lysate was assessed as described above. Means and standard deviations for 4 independent experiments are shown. RLU, relative light units.

    Techniques Used: Inhibition, Luciferase, Activity Assay, Transfection

    Analysis of cholesterol and NS5A abundances and their colocalization upon U18666A or Ro 48-8071 treatment. (A) Huh7/Lunet cells were transfected with the HCV full-length genome Jc1. After 24 h, cells were treated with the given drug concentrations for 48 h, and after fixation, free cholesterol (filipin; green), NS5A (red), and the lysosomal marker LAMP1 (blue) were visualized. White arrows indicate NS5A either colocalizing with filipin (control cells) or not (drug-treated cells). Representative images of cells treated with either 1.25 μM U18666A, 2.5 μM Ro 48-8071, or water (control) are shown. The signal intensity of filipin (integrated density, in arbitrary units [AU]) (B), the NS5A-filipin signal overlap (Manders overlap coefficient) (C), and the NS5A signal intensity (integrated density, in arbitrary units [AU]) (D) were determined for cells from the experiment shown for panel A. Uninfected and untreated cells are indicated by “no virus.” Results for two independent experiments for which at least 10 cells were analyzed are shown. Red bars indicate the medians. Each circle represents the result obtained for a single cell. (E) Representative immunoblot showing the protein levels of NS5A and beta actin under the corresponding conditions analyzed for panels A and B. The numbers on the bottom indicate the NS5A/beta actin signal ratio for two or three independent experiments. Note that NS5A abundance was not significantly affected at any drug concentration used. ***, P
    Figure Legend Snippet: Analysis of cholesterol and NS5A abundances and their colocalization upon U18666A or Ro 48-8071 treatment. (A) Huh7/Lunet cells were transfected with the HCV full-length genome Jc1. After 24 h, cells were treated with the given drug concentrations for 48 h, and after fixation, free cholesterol (filipin; green), NS5A (red), and the lysosomal marker LAMP1 (blue) were visualized. White arrows indicate NS5A either colocalizing with filipin (control cells) or not (drug-treated cells). Representative images of cells treated with either 1.25 μM U18666A, 2.5 μM Ro 48-8071, or water (control) are shown. The signal intensity of filipin (integrated density, in arbitrary units [AU]) (B), the NS5A-filipin signal overlap (Manders overlap coefficient) (C), and the NS5A signal intensity (integrated density, in arbitrary units [AU]) (D) were determined for cells from the experiment shown for panel A. Uninfected and untreated cells are indicated by “no virus.” Results for two independent experiments for which at least 10 cells were analyzed are shown. Red bars indicate the medians. Each circle represents the result obtained for a single cell. (E) Representative immunoblot showing the protein levels of NS5A and beta actin under the corresponding conditions analyzed for panels A and B. The numbers on the bottom indicate the NS5A/beta actin signal ratio for two or three independent experiments. Note that NS5A abundance was not significantly affected at any drug concentration used. ***, P

    Techniques Used: Transfection, Marker, Concentration Assay

    Effect of altered subcellular cholesterol distribution on morphology of the membranous web. (A) The replicon cells specified at the top were treated for 24 h with 0.625 μM U18666A, 0.625 μM Ro 48-8071, or water. Thereafter, the medium was replaced with fresh medium containing either water or the given drug in addition to either DMSO or 1 nM OSW-1. Cells were cultured for an additional 24 h and lysed for measurements of viral RNA replication by firefly luciferase assay (A) and cell viability (B). The results for two independent experiments are shown and are presented as percentages of the untreated control values. (C) Huh7/Lunet cells were electroporated with in vitro transcripts of the subgenomic replicon encoding mCherry-tagged NS5A. At 4 h postelectroporation, cells were treated with U18666A or Ro 48-8071 or left untreated, and 42 h later, cells were fixed and processed for electron microscopy analysis. Representative images are shown, with white boxes highlighting the areas shown as enlargements in the adjacent insets. White arrows indicate double-membrane vesicles. (D) DMV diameters were determined for untreated cells or cells treated with 1.25 μM or 0.625 μM U18666A or 1.25 μM Ro 48-8071. The results of a representative experiment are shown. At least 10 cell profiles were analyzed per condition. Each horizontal line indicates the median diameter, which is given numerically above each column. (E) Summary of the results of 2 or 3 independent experiments. The median DMV diameter for each experiment is indicated by a circle, while the black line represents the overall median. (F) Immunoblot showing NS5A abundances in lysates of cells corresponding to panel C; beta actin served as a loading control and for normalization. Numbers below the lanes are the ratios of NS5A and beta actin signals. ***, P
    Figure Legend Snippet: Effect of altered subcellular cholesterol distribution on morphology of the membranous web. (A) The replicon cells specified at the top were treated for 24 h with 0.625 μM U18666A, 0.625 μM Ro 48-8071, or water. Thereafter, the medium was replaced with fresh medium containing either water or the given drug in addition to either DMSO or 1 nM OSW-1. Cells were cultured for an additional 24 h and lysed for measurements of viral RNA replication by firefly luciferase assay (A) and cell viability (B). The results for two independent experiments are shown and are presented as percentages of the untreated control values. (C) Huh7/Lunet cells were electroporated with in vitro transcripts of the subgenomic replicon encoding mCherry-tagged NS5A. At 4 h postelectroporation, cells were treated with U18666A or Ro 48-8071 or left untreated, and 42 h later, cells were fixed and processed for electron microscopy analysis. Representative images are shown, with white boxes highlighting the areas shown as enlargements in the adjacent insets. White arrows indicate double-membrane vesicles. (D) DMV diameters were determined for untreated cells or cells treated with 1.25 μM or 0.625 μM U18666A or 1.25 μM Ro 48-8071. The results of a representative experiment are shown. At least 10 cell profiles were analyzed per condition. Each horizontal line indicates the median diameter, which is given numerically above each column. (E) Summary of the results of 2 or 3 independent experiments. The median DMV diameter for each experiment is indicated by a circle, while the black line represents the overall median. (F) Immunoblot showing NS5A abundances in lysates of cells corresponding to panel C; beta actin served as a loading control and for normalization. Numbers below the lanes are the ratios of NS5A and beta actin signals. ***, P

    Techniques Used: Cell Culture, Luciferase, In Vitro, Electron Microscopy

    NPC1-mediated cholesterol export at late endosome-lysosome–ER contact sites is important for the integrity of the membranous web in HCV-infected cells. Huh7/Lunet cells were electroporated with full-length RNA of the HCV genome Jc1. At 4 h postelectroporation, cells were treated with 1.25 μM U18666A or left untreated, and 42 h later, cells were fixed and processed for electron microscopy analysis. (A) Representative images, with white boxes highlighting the areas shown in the adjacent insets. White arrows indicate double-membrane vesicles (DMVs). (B and C) DMV diameters were determined for untreated cells or cells treated with 1.25 μM U18666A. Quantifications for two experiments are shown in panels B and C. At least 10 cells were analyzed per condition. Each horizontal line indicates the median, which is given numerically above each column. (D) Representative immunoblot showing NS5A and beta actin abundances in cell lysates corresponding to panel A. The numbers indicate the NS5A/beta actin signal ratios determined for two independent experiments. (E) Representative images showing DMVs with regular (white arrows) or aberrant (black arrows) morphology. (F) Quantification of the ratio of DMVs with regular morphology to DMVs with an aberrant shape. Each dot indicates the ratio determined for a single cell. The results of two independent experiments are shown. Each horizontal line indicates the median. ***, P
    Figure Legend Snippet: NPC1-mediated cholesterol export at late endosome-lysosome–ER contact sites is important for the integrity of the membranous web in HCV-infected cells. Huh7/Lunet cells were electroporated with full-length RNA of the HCV genome Jc1. At 4 h postelectroporation, cells were treated with 1.25 μM U18666A or left untreated, and 42 h later, cells were fixed and processed for electron microscopy analysis. (A) Representative images, with white boxes highlighting the areas shown in the adjacent insets. White arrows indicate double-membrane vesicles (DMVs). (B and C) DMV diameters were determined for untreated cells or cells treated with 1.25 μM U18666A. Quantifications for two experiments are shown in panels B and C. At least 10 cells were analyzed per condition. Each horizontal line indicates the median, which is given numerically above each column. (D) Representative immunoblot showing NS5A and beta actin abundances in cell lysates corresponding to panel A. The numbers indicate the NS5A/beta actin signal ratios determined for two independent experiments. (E) Representative images showing DMVs with regular (white arrows) or aberrant (black arrows) morphology. (F) Quantification of the ratio of DMVs with regular morphology to DMVs with an aberrant shape. Each dot indicates the ratio determined for a single cell. The results of two independent experiments are shown. Each horizontal line indicates the median. ***, P

    Techniques Used: Infection, Electron Microscopy

    Knockdown of NPC1 or its pharmacological inhibition alters the distribution of endogenous unesterified cholesterol. (A) Huh7/Lunet cells were transfected with the HCV full-length genome Jc1, and 4 h later, cells were transduced with a lentivirus (MOI = 4) encoding NPC1- or PI4KIIIA-specific shRNAs or a nontargeting shRNA (shNT) serving as a control. Sixty-eight hours later, cells were fixed, and free cholesterol (filipin; green) and NS5A (red) were visualized by indirect immunolabeling. The left panels show merged images, while the adjacent images display the filipin signal for each of three different cells. (B) Huh7/Lunet cells were transfected with the HCV full-length genome Jc1, and 24 h later, cells were treated with U18666A or Ro 48-8061 for 48 h. Free cholesterol (filipin; green), NS5A (red), and the lysosomal marker LAMP1 (blue) are shown. White arrows indicate NS5A either colocalizing with filipin (control) or not (drug-treated cells). (C) Quantification of the volumes of filipin-positive structures in the cells shown in panels A and B. At least 200 structures in 10 to 13 cells were analyzed. The red bars and numbers indicate the medians. ***, P
    Figure Legend Snippet: Knockdown of NPC1 or its pharmacological inhibition alters the distribution of endogenous unesterified cholesterol. (A) Huh7/Lunet cells were transfected with the HCV full-length genome Jc1, and 4 h later, cells were transduced with a lentivirus (MOI = 4) encoding NPC1- or PI4KIIIA-specific shRNAs or a nontargeting shRNA (shNT) serving as a control. Sixty-eight hours later, cells were fixed, and free cholesterol (filipin; green) and NS5A (red) were visualized by indirect immunolabeling. The left panels show merged images, while the adjacent images display the filipin signal for each of three different cells. (B) Huh7/Lunet cells were transfected with the HCV full-length genome Jc1, and 24 h later, cells were treated with U18666A or Ro 48-8061 for 48 h. Free cholesterol (filipin; green), NS5A (red), and the lysosomal marker LAMP1 (blue) are shown. White arrows indicate NS5A either colocalizing with filipin (control) or not (drug-treated cells). (C) Quantification of the volumes of filipin-positive structures in the cells shown in panels A and B. At least 200 structures in 10 to 13 cells were analyzed. The red bars and numbers indicate the medians. ***, P

    Techniques Used: Inhibition, Transfection, Transduction, shRNA, Immunolabeling, Marker

    37) 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

    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 "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

    40) 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

    41) 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

    42) 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

    43) 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

    44) 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

    45) Product Images from "Mutation of NgBR, a subunit of cis-prenyltransferase, causes a congenial disorder of glycosylation"

    Article Title: Mutation of NgBR, a subunit of cis-prenyltransferase, causes a congenial disorder of glycosylation

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2014.06.016

    NgBR R290H mutation causes defects in cellular cholesterol trafficking and the dolichol biosynthesis pathway ( A ) Filipin staining and quantitative representation for human dermal fibroblast cells from patients (II.3 and II.4). U18666A was used as a positive control for inhibition of cholesterol trafficking. ( B ) Microsomal cis-PTase activity using isolated membrane from fibroblasts. Compare to wild type, less than 20% of activity was detected in the patient cells. ( C ) [2- 3 H]-mannose-labeling of proteins. Cells were incubated with [2- 3 H]-mannose for 4hrs and TCA precipitated proteins were counted by scintillation. Tunicamycin (Tm) treatment was used as a control for loss of [2- 3 H]-mannose incorporation into newly synthesized proteins. *p
    Figure Legend Snippet: NgBR R290H mutation causes defects in cellular cholesterol trafficking and the dolichol biosynthesis pathway ( A ) Filipin staining and quantitative representation for human dermal fibroblast cells from patients (II.3 and II.4). U18666A was used as a positive control for inhibition of cholesterol trafficking. ( B ) Microsomal cis-PTase activity using isolated membrane from fibroblasts. Compare to wild type, less than 20% of activity was detected in the patient cells. ( C ) [2- 3 H]-mannose-labeling of proteins. Cells were incubated with [2- 3 H]-mannose for 4hrs and TCA precipitated proteins were counted by scintillation. Tunicamycin (Tm) treatment was used as a control for loss of [2- 3 H]-mannose incorporation into newly synthesized proteins. *p

    Techniques Used: Mutagenesis, Staining, Positive Control, Inhibition, Activity Assay, Isolation, Labeling, Incubation, Synthesized

    Characterization of NgBR knockout mouse embryos and fibroblasts ( A ) Genotype obtained from the progeny of heterozygous mating. No NgBR Δ/Δ embryo was detected. ( B ) Embryo resorption frequencies during post-implantation development. Resorption sites were apparent at E7.5 among ∼25% decidua. ( C ) Representative decidua of E 7.5 embryo resorption sites analyzed. Decidua were obtained from NgBR Δ/+ x NgBR Δ/+ breeding. Decidua with embryo contained normally developed E7.5 embryo (insert). Presumptive NgBR Δ/Δ decidua exhibit implanted site for embryo without evident embryonic material (arrowhead). ( D ) Filipin staining and quantitative representation for mouse embryonic fibroblast. Filipin staining was performed 48hrs after Lenti-NgBR transduction into NgBR iKO MEF cells. U18666A was used as a positive control for inhibition of cholesterol trafficking. ( E ) Microsomal cis-PTase activity assay for NgBR iKO MEF. 83% reduced enzyme activity was detected in NgBR iKO MEF compare to control. ( F ) [2- 3 H]-mannose-labeling of proteins in mouse embryonic fibroblasts. Tunicamycin (Tm) treatment was used as a control. ( G ) Statin sensitivity measured by MTT assay. Cell viability was determined by MTT assay after 16 h exposure with various concentration of lovastatin (1 to 80 μM). Cell viability was calculated by following equation: MTT OD value of treated sample/MTT OD value of non-treated sample. ( H .
    Figure Legend Snippet: Characterization of NgBR knockout mouse embryos and fibroblasts ( A ) Genotype obtained from the progeny of heterozygous mating. No NgBR Δ/Δ embryo was detected. ( B ) Embryo resorption frequencies during post-implantation development. Resorption sites were apparent at E7.5 among ∼25% decidua. ( C ) Representative decidua of E 7.5 embryo resorption sites analyzed. Decidua were obtained from NgBR Δ/+ x NgBR Δ/+ breeding. Decidua with embryo contained normally developed E7.5 embryo (insert). Presumptive NgBR Δ/Δ decidua exhibit implanted site for embryo without evident embryonic material (arrowhead). ( D ) Filipin staining and quantitative representation for mouse embryonic fibroblast. Filipin staining was performed 48hrs after Lenti-NgBR transduction into NgBR iKO MEF cells. U18666A was used as a positive control for inhibition of cholesterol trafficking. ( E ) Microsomal cis-PTase activity assay for NgBR iKO MEF. 83% reduced enzyme activity was detected in NgBR iKO MEF compare to control. ( F ) [2- 3 H]-mannose-labeling of proteins in mouse embryonic fibroblasts. Tunicamycin (Tm) treatment was used as a control. ( G ) Statin sensitivity measured by MTT assay. Cell viability was determined by MTT assay after 16 h exposure with various concentration of lovastatin (1 to 80 μM). Cell viability was calculated by following equation: MTT OD value of treated sample/MTT OD value of non-treated sample. ( H .

    Techniques Used: Knock-Out, Staining, Transduction, Positive Control, Inhibition, Activity Assay, Labeling, MTT Assay, Concentration Assay

    46) Product Images from "Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells"

    Article Title: Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106516

    Co-localization of PrP Sc with Snx1. ScN2a-3-22L cells grown on a chambered coverglass for 48 h were incubated with 7.5 µg/ml mAb 44B1, 10 µg/ml PPS, 10 µM CPZ, or 5 µM U18666A or without an anti-prion compound for 2 h. The cells were subjected to PrP Sc -specific staining with rIgG132-EGFP and immunostaining for Snx1. Nuclei were counterstained with DAPI. The leftmost column presents a lower-magnification merged image of PrP Sc (green), Snx1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows denote representative examples of co-localization of PrP Sc with Snx1. Scale bars: 10 µm.
    Figure Legend Snippet: Co-localization of PrP Sc with Snx1. ScN2a-3-22L cells grown on a chambered coverglass for 48 h were incubated with 7.5 µg/ml mAb 44B1, 10 µg/ml PPS, 10 µM CPZ, or 5 µM U18666A or without an anti-prion compound for 2 h. The cells were subjected to PrP Sc -specific staining with rIgG132-EGFP and immunostaining for Snx1. Nuclei were counterstained with DAPI. The leftmost column presents a lower-magnification merged image of PrP Sc (green), Snx1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows denote representative examples of co-localization of PrP Sc with Snx1. Scale bars: 10 µm.

    Techniques Used: Incubation, Staining, Immunostaining

    Influence of anti-prion compounds on the intracellular PrP Sc distribution. ScN2a-3-22L cells grown on a chambered coverglass for 48 h were cultured with or without an anti-prion compound at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6, 24, or 48 h. The cells were subjected to PrP Sc -specific detection by direct immunostaining with rIgG132-EGFP. The cell nuclei were counterstained with DAPI. The panels show the merged images of PrP Sc (green) and nuclei (blue). Scale bars: 10 µm.
    Figure Legend Snippet: Influence of anti-prion compounds on the intracellular PrP Sc distribution. ScN2a-3-22L cells grown on a chambered coverglass for 48 h were cultured with or without an anti-prion compound at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6, 24, or 48 h. The cells were subjected to PrP Sc -specific detection by direct immunostaining with rIgG132-EGFP. The cell nuclei were counterstained with DAPI. The panels show the merged images of PrP Sc (green) and nuclei (blue). Scale bars: 10 µm.

    Techniques Used: Cell Culture, Immunostaining

    Influence of anti-prion compounds on PrP C levels. N2a-3 cells were cultured with an anti-prion compound at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6–72 h and subjected to dot-blotting for the detection of PrP C or GAPDH for endogenous control. Representative dot-blot images are shown on the left, and the graph on the right shows the PrP C levels relative to that of 72-h mock-treated cells. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between the cells treated with each anti-prion compound and mock-treated control cells at the same time point (Student’s t- test, p
    Figure Legend Snippet: Influence of anti-prion compounds on PrP C levels. N2a-3 cells were cultured with an anti-prion compound at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6–72 h and subjected to dot-blotting for the detection of PrP C or GAPDH for endogenous control. Representative dot-blot images are shown on the left, and the graph on the right shows the PrP C levels relative to that of 72-h mock-treated cells. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between the cells treated with each anti-prion compound and mock-treated control cells at the same time point (Student’s t- test, p

    Techniques Used: Cell Culture, Dot Blot

    Influence of anti-prion compounds on the amount of PrP-res. (A) ScN2a-3-22L cells grown on 12-well plates were cultured in the presence or absence of mAb 44B1, PPS, CPZ, or U18666A at the indicated concentration for 72 h. The samples were subjected to immunoblotting and dot-blotting for PrP-res detection or β-actin detection for endogenous control. Representative blots for each compound are shown on the left. The graph on the right shows PrP-res levels relative to the control samples. The means and standard deviations (SDs) of four independent experiments (PrP-res was detected by dot-blotting) are indicated. Graphs on the upper right show the logistic curve fitted to the data of PrP-res levels by dot-blotting (B) ScN2a-3-22L cells were cultured with anti-prion compounds at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for the indicated time and subjected to dot-blotting for PrP-res. Representative dot-blotting is shown on the left, and the graph on the right shows the levels of PrP-res relative to the samples from ScN2a-3-22L cells cultured without anti-prion compounds for 72 h. The means and SDs of four independent experiments are depicted. Asterisks indicate a significant difference compared with the control at the same time point (Student’s t- test, p
    Figure Legend Snippet: Influence of anti-prion compounds on the amount of PrP-res. (A) ScN2a-3-22L cells grown on 12-well plates were cultured in the presence or absence of mAb 44B1, PPS, CPZ, or U18666A at the indicated concentration for 72 h. The samples were subjected to immunoblotting and dot-blotting for PrP-res detection or β-actin detection for endogenous control. Representative blots for each compound are shown on the left. The graph on the right shows PrP-res levels relative to the control samples. The means and standard deviations (SDs) of four independent experiments (PrP-res was detected by dot-blotting) are indicated. Graphs on the upper right show the logistic curve fitted to the data of PrP-res levels by dot-blotting (B) ScN2a-3-22L cells were cultured with anti-prion compounds at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for the indicated time and subjected to dot-blotting for PrP-res. Representative dot-blotting is shown on the left, and the graph on the right shows the levels of PrP-res relative to the samples from ScN2a-3-22L cells cultured without anti-prion compounds for 72 h. The means and SDs of four independent experiments are depicted. Asterisks indicate a significant difference compared with the control at the same time point (Student’s t- test, p

    Techniques Used: Cell Culture, Concentration Assay

    Co-localization of PrP Sc or PrP C with EEA1. ScN2a-3-22L cells were cultured with 7.5 µg/ml mAb 44B1, 10 µg/ml PPS, 10 µM CPZ or 5 µM U18666A or without an anti-prion compound for 6 h. The cells were subjected to direct immunostaining of PrP C and PrP Sc with 31C6-Af555 and rIgG132-EGFP, respectively and subsequently to immunostaining for EEA1 and nuclei. The leftmost column shows a lower-magnification merged image of PrP Sc (green), PrP C (cyan), EEA1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows or arrowheads denote representative examples of the co-localization of PrP Sc with EEA1 or PrP C with EEA1, respectively. Scale bars: 10 µm.
    Figure Legend Snippet: Co-localization of PrP Sc or PrP C with EEA1. ScN2a-3-22L cells were cultured with 7.5 µg/ml mAb 44B1, 10 µg/ml PPS, 10 µM CPZ or 5 µM U18666A or without an anti-prion compound for 6 h. The cells were subjected to direct immunostaining of PrP C and PrP Sc with 31C6-Af555 and rIgG132-EGFP, respectively and subsequently to immunostaining for EEA1 and nuclei. The leftmost column shows a lower-magnification merged image of PrP Sc (green), PrP C (cyan), EEA1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows or arrowheads denote representative examples of the co-localization of PrP Sc with EEA1 or PrP C with EEA1, respectively. Scale bars: 10 µm.

    Techniques Used: Cell Culture, Immunostaining

    Degradation of LDL in cells treated with anti-prion compounds. ScN2a-3-22L cells were incubated with Alexa Fluor 488-conjugated LDL (4 µg/ml) for 6 h. After the incubation, the cells were cultured in the presence or absence of 0.4 µg/ml mAb 44B1, 0.1 µg/ml PPS, 10 µg/ml CPZ, or 5 µM U18666A for 6–48 h. The cells were subjected to immunostaining of Lamp1 and staining of the cell nuclei with DAPI. Z-series of the images were acquired at 0.8-µm steps from the top to the bottom of the cells in the area. (A) Localization of LDL. The panel shows the representative images of the signals of LDL (green), Lamp1 (red) and nuclei (blue) in cells treated with the indicated anti-prion compound for 24 h. The merged images of LDL and nuclei are shown on the left, those of Lamp1 and nuclei are shown in the middle, and those of LDL, Lamp1, and nuclei are shown on the right. The rightmost column presents the higher-magnification images of the boxed regions in the second right column. Scale bars: 10 µm. (B) Intensity of LDL in Lamp1-positive vesicles. The graph shows the values of the fluorescent intensities of LDL in Lamp1-positive vesicles per cell relative to those of LDL in Lamp1-positive vesicles per cell in mock-treated control cells for 6 h. The means and SDs of the value acquired in five view fields are shown. Asterisks indicate a significant difference compared with the control at the same time point (Student’s t- test, p
    Figure Legend Snippet: Degradation of LDL in cells treated with anti-prion compounds. ScN2a-3-22L cells were incubated with Alexa Fluor 488-conjugated LDL (4 µg/ml) for 6 h. After the incubation, the cells were cultured in the presence or absence of 0.4 µg/ml mAb 44B1, 0.1 µg/ml PPS, 10 µg/ml CPZ, or 5 µM U18666A for 6–48 h. The cells were subjected to immunostaining of Lamp1 and staining of the cell nuclei with DAPI. Z-series of the images were acquired at 0.8-µm steps from the top to the bottom of the cells in the area. (A) Localization of LDL. The panel shows the representative images of the signals of LDL (green), Lamp1 (red) and nuclei (blue) in cells treated with the indicated anti-prion compound for 24 h. The merged images of LDL and nuclei are shown on the left, those of Lamp1 and nuclei are shown in the middle, and those of LDL, Lamp1, and nuclei are shown on the right. The rightmost column presents the higher-magnification images of the boxed regions in the second right column. Scale bars: 10 µm. (B) Intensity of LDL in Lamp1-positive vesicles. The graph shows the values of the fluorescent intensities of LDL in Lamp1-positive vesicles per cell relative to those of LDL in Lamp1-positive vesicles per cell in mock-treated control cells for 6 h. The means and SDs of the value acquired in five view fields are shown. Asterisks indicate a significant difference compared with the control at the same time point (Student’s t- test, p

    Techniques Used: Incubation, Cell Culture, Immunostaining, Staining

    Influence of anti-prion compounds on the localization of PrP Sc and PrP C . N2a-3 cells or ScN2a-3-22L cells were cultured with or without an anti-prion compound at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6 h. The cells were fixed and stained with 31C6-Af555 to detect PrP C , and subsequently subjected to PrP Sc -specific detection with rIgG132-EGFP. The cell nuclei were counterstained with DAPI. The merged images of PrP Sc (green) and nuclei (blue) are shown on the left, those of PrP C (red) and nuclei are shown in the middle, and those of PrP Sc , PrP C , and nuclei are shown on the right. Scale bars: 10 µm.
    Figure Legend Snippet: Influence of anti-prion compounds on the localization of PrP Sc and PrP C . N2a-3 cells or ScN2a-3-22L cells were cultured with or without an anti-prion compound at the EC 65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6 h. The cells were fixed and stained with 31C6-Af555 to detect PrP C , and subsequently subjected to PrP Sc -specific detection with rIgG132-EGFP. The cell nuclei were counterstained with DAPI. The merged images of PrP Sc (green) and nuclei (blue) are shown on the left, those of PrP C (red) and nuclei are shown in the middle, and those of PrP Sc , PrP C , and nuclei are shown on the right. Scale bars: 10 µm.

    Techniques Used: Cell Culture, Staining

    Effect of lysosomal hydrolysis inhibition on the decrease of PrP Sc levels induced by CPZ or U18666A treatment. (A) Immunoblot analysis of PrP-res and cathepsin D. ScN2a-3-22L cells were cultured with 10 µM CPZ or 5 µM U18666A or without compounds for 24 h. Subsequently, monensin (Mon) or bafilomycin A1 (BafA1) was added to the culture at a final concentration of 100 or 5 nM, respectively. Following an additional incubation for 36 h with or without Mon or BafA1, the cells were subjected to immunoblotting for PrP-res, cathepsin D, or β-actin. Representative immunoblot images are shown on the left. The bracket in the immunoblot of cathepsin D denotes the pro- and/or intermediate forms of cathepsin D (Pro/Int). The arrowhead denotes the mature form of cathepsin D (M). The upper right graph shows the levels of PrP-res relative to the control. The lower right graph shows the ratio of mature to pro−/intermediate forms of cathepsin D. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between Mon- or BafA1-treated samples and untreated samples (non-treated) (Student’s t- test, p
    Figure Legend Snippet: Effect of lysosomal hydrolysis inhibition on the decrease of PrP Sc levels induced by CPZ or U18666A treatment. (A) Immunoblot analysis of PrP-res and cathepsin D. ScN2a-3-22L cells were cultured with 10 µM CPZ or 5 µM U18666A or without compounds for 24 h. Subsequently, monensin (Mon) or bafilomycin A1 (BafA1) was added to the culture at a final concentration of 100 or 5 nM, respectively. Following an additional incubation for 36 h with or without Mon or BafA1, the cells were subjected to immunoblotting for PrP-res, cathepsin D, or β-actin. Representative immunoblot images are shown on the left. The bracket in the immunoblot of cathepsin D denotes the pro- and/or intermediate forms of cathepsin D (Pro/Int). The arrowhead denotes the mature form of cathepsin D (M). The upper right graph shows the levels of PrP-res relative to the control. The lower right graph shows the ratio of mature to pro−/intermediate forms of cathepsin D. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between Mon- or BafA1-treated samples and untreated samples (non-treated) (Student’s t- test, p

    Techniques Used: Inhibition, Cell Culture, Concentration Assay, Incubation

    Induction of autophagy by CPZ or U18666A treatment. ScN2a-3-22L cells were cultured under the same conditions as described in Figure 6 . The cells were subjected to immunostaining for LC3 and Lamp1 and counterstained with DAPI. The leftmost column shows the lower-magnification merged image of LC3 (green), Lamp1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Scale bars: 10 µm.
    Figure Legend Snippet: Induction of autophagy by CPZ or U18666A treatment. ScN2a-3-22L cells were cultured under the same conditions as described in Figure 6 . The cells were subjected to immunostaining for LC3 and Lamp1 and counterstained with DAPI. The leftmost column shows the lower-magnification merged image of LC3 (green), Lamp1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Scale bars: 10 µm.

    Techniques Used: Cell Culture, Immunostaining

    Fate of PrP Sc after treatment with CPZ or U18666A. ScN2a-3-22L cells were cultured with 10 µg/ml PPS, 10 µM CPZ or 5 µM U18666A or without an anti-prion compound for 24 or 48 h. The cells were subjected to the PrP Sc -specific indirect immunostaining of PrP Sc with mAb 132 and immunostaining for Lamp1 and nuclei. (A) Localization of PrP Sc . The left columns show a lower-magnification merged image of PrP Sc (green), Lamp1 (red), and nuclei (blue). The high-magnification images of the boxed regions are shown on the right. Arrows indicate representative co-localization of PrP Sc with Lamp1. Arrows with asterisks indicate swollen Lamp1-positive vesicles positive for PrP Sc . Scale bars: 10 µm. (B) Intensity of PrP Sc in Lamp1-positive vesicles. The graph represents the values of the fluorescent intensities of PrP Sc in Lamp1-positive vesicles per cell relative to those of PrP Sc in Lamp1-positive vesicles per cell in mock-treated cells after 24 h of treatment. The means and SDs of the value acquired in six view fields are shown. Double asterisks indicate a significant difference between cells treated for 24 and 48 h (Student’s t- test, p
    Figure Legend Snippet: Fate of PrP Sc after treatment with CPZ or U18666A. ScN2a-3-22L cells were cultured with 10 µg/ml PPS, 10 µM CPZ or 5 µM U18666A or without an anti-prion compound for 24 or 48 h. The cells were subjected to the PrP Sc -specific indirect immunostaining of PrP Sc with mAb 132 and immunostaining for Lamp1 and nuclei. (A) Localization of PrP Sc . The left columns show a lower-magnification merged image of PrP Sc (green), Lamp1 (red), and nuclei (blue). The high-magnification images of the boxed regions are shown on the right. Arrows indicate representative co-localization of PrP Sc with Lamp1. Arrows with asterisks indicate swollen Lamp1-positive vesicles positive for PrP Sc . Scale bars: 10 µm. (B) Intensity of PrP Sc in Lamp1-positive vesicles. The graph represents the values of the fluorescent intensities of PrP Sc in Lamp1-positive vesicles per cell relative to those of PrP Sc in Lamp1-positive vesicles per cell in mock-treated cells after 24 h of treatment. The means and SDs of the value acquired in six view fields are shown. Double asterisks indicate a significant difference between cells treated for 24 and 48 h (Student’s t- test, p

    Techniques Used: Cell Culture, Immunostaining

    47) Product Images from "Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity"

    Article Title: Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2008.03.019

    Reversal of the effects of ceramide and SMases on cholesterol esterification by U18666A
    Figure Legend Snippet: Reversal of the effects of ceramide and SMases on cholesterol esterification by U18666A

    Techniques Used:

    Reversal of the effects of ceramide and SMases on HMGCR by U18666A
    Figure Legend Snippet: Reversal of the effects of ceramide and SMases on HMGCR by U18666A

    Techniques Used:

    48) Product Images from "25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes"

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.09.003

    Assessment of the antiviral activity of U18666A against HRV strains and assessment of its mechanism of action. Cells were treated for 20 h with increasing concentrations of U18666A and then infected with HRV Wa (A), WI61 (B), HRV 248 (C), DS-1 (D), and HRV 408 (E). Viral infections were detected as described in the Material and Methods section. The percentage infection was calculated by comparing treated and untreated wells. The results are means and SEM for triplicates.***p ANOVA
    Figure Legend Snippet: Assessment of the antiviral activity of U18666A against HRV strains and assessment of its mechanism of action. Cells were treated for 20 h with increasing concentrations of U18666A and then infected with HRV Wa (A), WI61 (B), HRV 248 (C), DS-1 (D), and HRV 408 (E). Viral infections were detected as described in the Material and Methods section. The percentage infection was calculated by comparing treated and untreated wells. The results are means and SEM for triplicates.***p ANOVA

    Techniques Used: Activity Assay, Infection

    Effect of 25HC or 27HC on intracellular localization of cholesterol. Positive control experiments were performed by treating cells with U18666A. Treated or untreated MA104 were fixed at 20 h post-treatment and intracellular cholesterol was stained with filipin (blue signal)and late endosomes with anti-Rab7 antibody (green signal). The inserts in the right panels show the merged signals.
    Figure Legend Snippet: Effect of 25HC or 27HC on intracellular localization of cholesterol. Positive control experiments were performed by treating cells with U18666A. Treated or untreated MA104 were fixed at 20 h post-treatment and intracellular cholesterol was stained with filipin (blue signal)and late endosomes with anti-Rab7 antibody (green signal). The inserts in the right panels show the merged signals.

    Techniques Used: Positive Control, Staining

    49) 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

    50) Product Images from "The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution"

    Article Title: The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709123

    RELCH, OSBP, and Rab11 depletion results in less cholesterol accumulation in the TGN and ER. (A and B) The homogenates from the shRNA-expressing HeLa cells were fractionated using a Histodenz step density gradient. (A) The fractions were immunoblotted (IB) with antibodies against TGN46, calnexin, and Lamp2. (B) Percentages of cholesterol (µg/mg protein) in the TGN (fractions 4–6), LEs/lysosomes (LE/LY; 7–9), or ER (10 and 11) in the total fractions are shown in the bar graph. (C) HeLa cells were treated with 2 µg/ml U18666A for 16 h and stained with Filipin. Bar, 20 µm. (D–G) Immunoisolation of ER, TGN, and LE/lysosome from the PNS derived from the U18666A-treated (D and E) or RELCH-, Rab11a/b-, and OSBP-depleted cells by shRNAs (F and G). ER, TGN, and LE/lysosome membranes were isolated using calnexin, TGN46, and Lamp1 antibodies, respectively. (D and F) The isolated samples were immunoblotted with calnexin, TGN46, and Lamp2 antibodies. (E and G) Quantification of the cholesterol content in the isolated membranes (relative to the control samples). Data are expressed as means ± SEM from at least three independent experiments. *, P
    Figure Legend Snippet: RELCH, OSBP, and Rab11 depletion results in less cholesterol accumulation in the TGN and ER. (A and B) The homogenates from the shRNA-expressing HeLa cells were fractionated using a Histodenz step density gradient. (A) The fractions were immunoblotted (IB) with antibodies against TGN46, calnexin, and Lamp2. (B) Percentages of cholesterol (µg/mg protein) in the TGN (fractions 4–6), LEs/lysosomes (LE/LY; 7–9), or ER (10 and 11) in the total fractions are shown in the bar graph. (C) HeLa cells were treated with 2 µg/ml U18666A for 16 h and stained with Filipin. Bar, 20 µm. (D–G) Immunoisolation of ER, TGN, and LE/lysosome from the PNS derived from the U18666A-treated (D and E) or RELCH-, Rab11a/b-, and OSBP-depleted cells by shRNAs (F and G). ER, TGN, and LE/lysosome membranes were isolated using calnexin, TGN46, and Lamp1 antibodies, respectively. (D and F) The isolated samples were immunoblotted with calnexin, TGN46, and Lamp2 antibodies. (E and G) Quantification of the cholesterol content in the isolated membranes (relative to the control samples). Data are expressed as means ± SEM from at least three independent experiments. *, P

    Techniques Used: shRNA, Expressing, Staining, Derivative Assay, Isolation

    51) Product Images from "A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes"

    Article Title: A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200807186

    U18666A and bafilomycin induce accumulation of KRas in a Rab7/LBPA late endosomal compartment. (A) HA-KRas and GFP-Rab7–expressing cells were treated with EGF for 20 min and immunolabeled with an anti-HA (red channel) and anti-EEA1 (blue channel). Arrows indicate the colocalization of KRas and EEA1, and arrowheads point to Rab7-positive structures. (B) Localization of GFP-KRas in cells treated with U18666A. Arrows point to PM KRas labeling, and arrowheads show intracellular ringlike structures. (C) HA-KRas was coexpressed with GFP-Rab7 and treated with U18666A plus EGF for 45 min. Arrows show colocalization of HA-KRas with GFP-Rab7. (D–F) Localization of KRas in cells treated with 20 nM B-A1 overnight and with 100 ng/ml EGF for 45 min. Arrows indicate colocalization of HA-KRas and Rab7 (E) or GFP-KRas and LBPA (F) on enlarged endosomes. The white dotted contour in D indicates the cell perimeter, and the arrowheads point to ringlike endosomal structures. Bars: (A–D) 8 µm; (E and F) 2 µm.
    Figure Legend Snippet: U18666A and bafilomycin induce accumulation of KRas in a Rab7/LBPA late endosomal compartment. (A) HA-KRas and GFP-Rab7–expressing cells were treated with EGF for 20 min and immunolabeled with an anti-HA (red channel) and anti-EEA1 (blue channel). Arrows indicate the colocalization of KRas and EEA1, and arrowheads point to Rab7-positive structures. (B) Localization of GFP-KRas in cells treated with U18666A. Arrows point to PM KRas labeling, and arrowheads show intracellular ringlike structures. (C) HA-KRas was coexpressed with GFP-Rab7 and treated with U18666A plus EGF for 45 min. Arrows show colocalization of HA-KRas with GFP-Rab7. (D–F) Localization of KRas in cells treated with 20 nM B-A1 overnight and with 100 ng/ml EGF for 45 min. Arrows indicate colocalization of HA-KRas and Rab7 (E) or GFP-KRas and LBPA (F) on enlarged endosomes. The white dotted contour in D indicates the cell perimeter, and the arrowheads point to ringlike endosomal structures. Bars: (A–D) 8 µm; (E and F) 2 µm.

    Techniques Used: Expressing, Immunolabeling, Labeling

    52) Product Images from "Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane"

    Article Title: Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1005095

    QuASIMoDOH reveals changes in lipid distribution patterns. (A) MEF cells stained for the lipid sphingomyelin using Lysenin. Images exhibit a characteristic clustered appearance of spots. (B) Zoomed in region indicated by the yellow rectangle in (A). (C) MEF cells were treated with 3 μg/mL U18666A for 18 h. (D) Zoomed in region indicated by the yellow rectangle in (C). (E-F) Upon drug treatment, the sphingomyelin distribution changes: clusters on the cell membrane appear to be smaller and more homogenous. The inhomogeneity measure reveals a shift to a more homogeneous distribution of Lysenin upon drug treatment (deviation from random significantly shifts from 0.36 to 0.18). Scale bar in (A and C): 5 μm. Scale bar in (B and D): 2 μm. Error bars represent the SEM. Analysis based on 49 cells for both untreated and U18666A treated cells. The average r 2 is 0.72 and 0.75 for untreated and drug-treated cells, respectively.
    Figure Legend Snippet: QuASIMoDOH reveals changes in lipid distribution patterns. (A) MEF cells stained for the lipid sphingomyelin using Lysenin. Images exhibit a characteristic clustered appearance of spots. (B) Zoomed in region indicated by the yellow rectangle in (A). (C) MEF cells were treated with 3 μg/mL U18666A for 18 h. (D) Zoomed in region indicated by the yellow rectangle in (C). (E-F) Upon drug treatment, the sphingomyelin distribution changes: clusters on the cell membrane appear to be smaller and more homogenous. The inhomogeneity measure reveals a shift to a more homogeneous distribution of Lysenin upon drug treatment (deviation from random significantly shifts from 0.36 to 0.18). Scale bar in (A and C): 5 μm. Scale bar in (B and D): 2 μm. Error bars represent the SEM. Analysis based on 49 cells for both untreated and U18666A treated cells. The average r 2 is 0.72 and 0.75 for untreated and drug-treated cells, respectively.

    Techniques Used: Staining

    53) 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

    54) Product Images from "Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium"

    Article Title: Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium

    Journal: Scientific Reports

    doi: 10.1038/srep41226

    SERMs reduce the cellular sphingosine. ( a ) Standard curves for quantification of Sph (0.1–100 ng/mL) with C17-Sph (10 ng/mL) as the internal standard. ( b) Measurement of changes in intracellular levels of Sph in HepG2 cells. Cells were treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h, then lipids were extracted and the levels of Sph were analyzed. Data are expressed as the means ± SEM (n = 3). Significant differences versus control group are presented by asterisks (*), *** P
    Figure Legend Snippet: SERMs reduce the cellular sphingosine. ( a ) Standard curves for quantification of Sph (0.1–100 ng/mL) with C17-Sph (10 ng/mL) as the internal standard. ( b) Measurement of changes in intracellular levels of Sph in HepG2 cells. Cells were treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h, then lipids were extracted and the levels of Sph were analyzed. Data are expressed as the means ± SEM (n = 3). Significant differences versus control group are presented by asterisks (*), *** P

    Techniques Used:

    Equal dosages of SERMs inhibit Ebola pseudovirion entry and induce cholesterol accumulation. ( a ) Dose-response inhibition of Ebola entry for tamoxifen, clomiphene and U18666a. Data are expressed as the means ± SEM (n = 3). ( b ) Dose-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. ( b ) Time-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. Black arrows indicate examples of cholesterol accumulation. ( d,e ) The quantification of cholesterol accumulation stained with filipin. Data are expressed as the means ± SEM (n ≥ 4). Significant differences versus control group (0 μM or 0 h) are presented by asterisks (*), * P
    Figure Legend Snippet: Equal dosages of SERMs inhibit Ebola pseudovirion entry and induce cholesterol accumulation. ( a ) Dose-response inhibition of Ebola entry for tamoxifen, clomiphene and U18666a. Data are expressed as the means ± SEM (n = 3). ( b ) Dose-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. ( b ) Time-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. Black arrows indicate examples of cholesterol accumulation. ( d,e ) The quantification of cholesterol accumulation stained with filipin. Data are expressed as the means ± SEM (n ≥ 4). Significant differences versus control group (0 μM or 0 h) are presented by asterisks (*), * P

    Techniques Used: Inhibition, Staining

    SERMs upregulate the endolysosomal calcium levels. ( a ) Representative images of the endolysosomal calcium release of HepG2 detected by Fluo8-AM calcium indicator at 1 s, 60 s, 90 s, 330 s, 390 s, and 420 s after incubation of 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( b ) The calcium release of HepG2 loaded with 1 μM TG at 60 s for 30 s and 200 μM GPN at 330 s for 60 s. HepG2 cells were pre-treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( c ) The quantification of calcium release induced by 200 μM GPN. Data are expressed as the means ± SEM (n > 20). Significant differences versus control group are presented by asterisks (*), *** P
    Figure Legend Snippet: SERMs upregulate the endolysosomal calcium levels. ( a ) Representative images of the endolysosomal calcium release of HepG2 detected by Fluo8-AM calcium indicator at 1 s, 60 s, 90 s, 330 s, 390 s, and 420 s after incubation of 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( b ) The calcium release of HepG2 loaded with 1 μM TG at 60 s for 30 s and 200 μM GPN at 330 s for 60 s. HepG2 cells were pre-treated with 10 μM tamoxifen, clomiphene or U18666a for 1 h. ( c ) The quantification of calcium release induced by 200 μM GPN. Data are expressed as the means ± SEM (n > 20). Significant differences versus control group are presented by asterisks (*), *** P

    Techniques Used: Incubation

    Proposed hypothesis of Ebola entry inhibition by CADs. Under normal conditions, Ebola is internalized by the NPC1+/TPC2+ endolysosome, in which the primed GP interacts with NPC1 domain C. The ASM and AC in the endolysosome then hydrolyze sphingomyelin to sphingosine. The elevated sphingosine induces endolysosomal calcium outflow through TPC1 and TPC2. Both the local calcium flux and the interaction of primed GP with NPC1 domain C induce conformational changes in the Ebola fusion peptide, triggering the membrane fusion during the Ebola infection. When treated with CADs that have selective anti-Ebola activities, including SERMs and U18666a, the CADs are protonated and trapped inside the acidic endolysosome. Due to their structural properties, these concentrated CADs induce the detachment of the ASM protein from membranes, which inactivates the ASM and in turn leads to a decrease in sphingosine. Without sufficient sphingosine, the endolysosome calcium outflow mediated by TPC1 and TPC2 are blocked, leading the endolysosome calcium accumulation. Without the calcium flux, there is no conformational change of the Ebola fusion peptide. Thus, there is neither membrane fusion nor Ebola infection.
    Figure Legend Snippet: Proposed hypothesis of Ebola entry inhibition by CADs. Under normal conditions, Ebola is internalized by the NPC1+/TPC2+ endolysosome, in which the primed GP interacts with NPC1 domain C. The ASM and AC in the endolysosome then hydrolyze sphingomyelin to sphingosine. The elevated sphingosine induces endolysosomal calcium outflow through TPC1 and TPC2. Both the local calcium flux and the interaction of primed GP with NPC1 domain C induce conformational changes in the Ebola fusion peptide, triggering the membrane fusion during the Ebola infection. When treated with CADs that have selective anti-Ebola activities, including SERMs and U18666a, the CADs are protonated and trapped inside the acidic endolysosome. Due to their structural properties, these concentrated CADs induce the detachment of the ASM protein from membranes, which inactivates the ASM and in turn leads to a decrease in sphingosine. Without sufficient sphingosine, the endolysosome calcium outflow mediated by TPC1 and TPC2 are blocked, leading the endolysosome calcium accumulation. Without the calcium flux, there is no conformational change of the Ebola fusion peptide. Thus, there is neither membrane fusion nor Ebola infection.

    Techniques Used: Inhibition, Infection

    55) Product Images from "Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking"

    Article Title: Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2009.07.003

    Loss of NgBR induces free cholesterol accumulation in cells (A) EA.hy926 cells or (B) NgBR +/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p
    Figure Legend Snippet: Loss of NgBR induces free cholesterol accumulation in cells (A) EA.hy926 cells or (B) NgBR +/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p

    Techniques Used: Incubation, Small Interfering RNA, Positive Control, Inhibition, Staining, Infection, Construct, Expressing, Transfection

    56) Product Images from "25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes"

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.09.003

    Assessment of the antiviral activity of U18666A against HRV strains and assessment of its mechanism of action. Cells were treated for 20 h with increasing concentrations of U18666A and then infected with HRV Wa (A), WI61 (B), HRV 248 (C), DS-1 (D), and HRV 408 (E). Viral infections were detected as described in the Material and Methods section. The percentage infection was calculated by comparing treated and untreated wells. The results are means and SEM for triplicates.***p ANOVA
    Figure Legend Snippet: Assessment of the antiviral activity of U18666A against HRV strains and assessment of its mechanism of action. Cells were treated for 20 h with increasing concentrations of U18666A and then infected with HRV Wa (A), WI61 (B), HRV 248 (C), DS-1 (D), and HRV 408 (E). Viral infections were detected as described in the Material and Methods section. The percentage infection was calculated by comparing treated and untreated wells. The results are means and SEM for triplicates.***p ANOVA

    Techniques Used: Activity Assay, Infection

    57) Product Images from "A mechanism for overcoming P-glycoprotein-mediated drug resistance: novel combination therapy that releases stored doxorubicin from lysosomes via lysosomal permeabilization using Dp44mT or DpC"

    Article Title: A mechanism for overcoming P-glycoprotein-mediated drug resistance: novel combination therapy that releases stored doxorubicin from lysosomes via lysosomal permeabilization using Dp44mT or DpC

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.381

    The cholesterol transport inhibitor, U18666A, prevents lysosomal-membrane permeabilization (LMP) following treatment with Dp44mT or DpC. Live cell immunofluorescence microscopy images using the following conditions: ( a ) KBV1 cells (+Pgp); ( b ) KBV1 cells with U18666A (2.3 μ g/ml); ( c ) KB31 cells (expressing extremely low Pgp levels) 24 and ( d ) KB31 cells with U18666A (2.3 μ g/ml). Cells were incubated for 30 min/37 °C with: (i) no treatment (untreated); (ii) Cu[Dp44mT] (30 μ M); or (iii) Cu[DpC] (15 μ M). Cells were then stained for 12 min/37 °C with acridine orange (AO; 5 μ M). The AO staining was quantified as red fluorescence intensity/cell using ImageJ software. *** P
    Figure Legend Snippet: The cholesterol transport inhibitor, U18666A, prevents lysosomal-membrane permeabilization (LMP) following treatment with Dp44mT or DpC. Live cell immunofluorescence microscopy images using the following conditions: ( a ) KBV1 cells (+Pgp); ( b ) KBV1 cells with U18666A (2.3 μ g/ml); ( c ) KB31 cells (expressing extremely low Pgp levels) 24 and ( d ) KB31 cells with U18666A (2.3 μ g/ml). Cells were incubated for 30 min/37 °C with: (i) no treatment (untreated); (ii) Cu[Dp44mT] (30 μ M); or (iii) Cu[DpC] (15 μ M). Cells were then stained for 12 min/37 °C with acridine orange (AO; 5 μ M). The AO staining was quantified as red fluorescence intensity/cell using ImageJ software. *** P

    Techniques Used: Immunofluorescence, Microscopy, Expressing, Incubation, Staining, Fluorescence, Software

    Inhibition of intracellular cholesterol transport by U18666A prevents drug synergy between DOX and the thiosemicarbazones, Dp44mT and DpC. ( a ) Cytotoxicity assays with the treatments: (i) DOX (72 h/37 °C), (ii) Dp44mT (24 h/37 °C) and (iii) DpC (24 h/37 °C), in the absence or presence of U18666A (2.3 μ g/ml). ( b ) Combination index (CI) values of the drug combination between: (i) DOX (72 h/37 °C) and Dp44mT (24 h/37 °C); or (ii) DOX (72 h/37 °C) and DpC (24 h/37 °C), as measured by the Chou-Talalay method. 25 , 29 The correlation between the CI values ( b iii and biv) and the cellular Pgp activity (as measured by Rh123 retention) was plotted using linear regression for: ( b iii) DOX (72 h/37 °C) and Dp44mT (24 h/37 °C); or ( b iv) DOX (72 h/37 °C) and DpC (24 h/37 °C). CI > 1 Antagonism, CI=1 Additive, CI
    Figure Legend Snippet: Inhibition of intracellular cholesterol transport by U18666A prevents drug synergy between DOX and the thiosemicarbazones, Dp44mT and DpC. ( a ) Cytotoxicity assays with the treatments: (i) DOX (72 h/37 °C), (ii) Dp44mT (24 h/37 °C) and (iii) DpC (24 h/37 °C), in the absence or presence of U18666A (2.3 μ g/ml). ( b ) Combination index (CI) values of the drug combination between: (i) DOX (72 h/37 °C) and Dp44mT (24 h/37 °C); or (ii) DOX (72 h/37 °C) and DpC (24 h/37 °C), as measured by the Chou-Talalay method. 25 , 29 The correlation between the CI values ( b iii and biv) and the cellular Pgp activity (as measured by Rh123 retention) was plotted using linear regression for: ( b iii) DOX (72 h/37 °C) and Dp44mT (24 h/37 °C); or ( b iv) DOX (72 h/37 °C) and DpC (24 h/37 °C). CI > 1 Antagonism, CI=1 Additive, CI

    Techniques Used: Inhibition, Activity Assay

    Schematic illustration of the synergistic interaction between Doxorubicin and Dp44mT/DpC. (1) Pgp on the plasma membrane actively pumps Pgp substrates out of cells. As part of endocytosis, the plasma membrane containing Pgp buds inwards to form early endosomes. (2) As a consequence of endocytosis, the topology of Pgp is inverted, and thus substrates are transported into the vesicle lumen. As the endosome matures into the lysosome, it becomes increasingly acidified. When a Pgp substrate, such as (3) Dp44mT/DpC, or (4) DOX, enters the cell, the drug is sequestered into the acidic lysosomes by Pgp-transport activity. 4 , 5 , 6 If the substrate is protonated at acidic pH (such as DOX and Dp44mT/DpC), it becomes trapped in lysosomes. 4 , 5 , 6 , 9 The trapping of protonated drugs prevents substrates reaching their molecular targets (e.g., the nucleus for DOX). 4 (3) However, once trapped in the lysosome, Dp44mT or DpC bind copper and redox cycle forming reactive oxygen species (ROS) that cause lysosomal-membrane permeabilization (LMP) and then apoptosis. 5 , 6 , 9 , 16 (5) When added in combination with DOX, Dp44mT or DpC redox cycle in lysosomes containing trapped DOX. (6) Dp44mT- or DpC-induced LMP causes the release of trapped DOX. (7) Then DOX redistributes to its molecular target, the nucleus. Notably, the Pgp-transport inhibitor, Ela, prevents entrance of Dp44mT or DpC into lysosomes, blocking LMP and the release of DOX to the nucleus. On the other hand, U18666A partially inhibits Pgp activity and also stabilizes the lysosomal membrane by cholesterol loading and prevents LMP mediated by Dp44mT or DpC, inhibiting lysosomal DOX release
    Figure Legend Snippet: Schematic illustration of the synergistic interaction between Doxorubicin and Dp44mT/DpC. (1) Pgp on the plasma membrane actively pumps Pgp substrates out of cells. As part of endocytosis, the plasma membrane containing Pgp buds inwards to form early endosomes. (2) As a consequence of endocytosis, the topology of Pgp is inverted, and thus substrates are transported into the vesicle lumen. As the endosome matures into the lysosome, it becomes increasingly acidified. When a Pgp substrate, such as (3) Dp44mT/DpC, or (4) DOX, enters the cell, the drug is sequestered into the acidic lysosomes by Pgp-transport activity. 4 , 5 , 6 If the substrate is protonated at acidic pH (such as DOX and Dp44mT/DpC), it becomes trapped in lysosomes. 4 , 5 , 6 , 9 The trapping of protonated drugs prevents substrates reaching their molecular targets (e.g., the nucleus for DOX). 4 (3) However, once trapped in the lysosome, Dp44mT or DpC bind copper and redox cycle forming reactive oxygen species (ROS) that cause lysosomal-membrane permeabilization (LMP) and then apoptosis. 5 , 6 , 9 , 16 (5) When added in combination with DOX, Dp44mT or DpC redox cycle in lysosomes containing trapped DOX. (6) Dp44mT- or DpC-induced LMP causes the release of trapped DOX. (7) Then DOX redistributes to its molecular target, the nucleus. Notably, the Pgp-transport inhibitor, Ela, prevents entrance of Dp44mT or DpC into lysosomes, blocking LMP and the release of DOX to the nucleus. On the other hand, U18666A partially inhibits Pgp activity and also stabilizes the lysosomal membrane by cholesterol loading and prevents LMP mediated by Dp44mT or DpC, inhibiting lysosomal DOX release

    Techniques Used: Activity Assay, Blocking Assay

    The cholesterol transport inhibitor, U18666A, prevents the release of DOX from LysoTracker Green-stained lysosomes following thiosemicarbazone treatment. Immunofluorescence microscopy images of KBV1 (+Pgp) cells treated with DOX (2 h/37 °C; 100 μ M), LysoTracker Green (40 min/37 °C; 100 nM) and 30 min/37 °C after either: ( a ) no treatment (Control), ( b ) Cu[Dp44mT] (30 μ M) or ( c ) Cu[DpC] (15 μ M), in the (i) absence of U18666A or (ii) presence of U18666A (2.3 μ g/ml). The overlap between DOX and LysoTracker Green (yellow merge; a – c ) is indicated by Mander's overlap coefficient ( R ). LysoTracker Green was quantified as fluorescence intensity/cell, while DOX was quantified as DOX fluorescence intensity co-distribution with LysoTracker Green or DAPI, using ImageJ software. ** P
    Figure Legend Snippet: The cholesterol transport inhibitor, U18666A, prevents the release of DOX from LysoTracker Green-stained lysosomes following thiosemicarbazone treatment. Immunofluorescence microscopy images of KBV1 (+Pgp) cells treated with DOX (2 h/37 °C; 100 μ M), LysoTracker Green (40 min/37 °C; 100 nM) and 30 min/37 °C after either: ( a ) no treatment (Control), ( b ) Cu[Dp44mT] (30 μ M) or ( c ) Cu[DpC] (15 μ M), in the (i) absence of U18666A or (ii) presence of U18666A (2.3 μ g/ml). The overlap between DOX and LysoTracker Green (yellow merge; a – c ) is indicated by Mander's overlap coefficient ( R ). LysoTracker Green was quantified as fluorescence intensity/cell, while DOX was quantified as DOX fluorescence intensity co-distribution with LysoTracker Green or DAPI, using ImageJ software. ** P

    Techniques Used: Staining, Immunofluorescence, Microscopy, Fluorescence, Software

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

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

    Journal: The EMBO Journal

    doi: 10.15252/embj.201695917

    Cholesterol staining with GFP‐D4 or filipin Plasma membrane cholesterol staining with the GFP‐D4 probe. Live HeLa/Ctrl cells were left untreated (a) or treated with MβCD (b) to remove plasma membrane cholesterol (10 mM in serum‐free medium; 30 min at 37°C), and incubated with GFP‐D4 prior to fixation and nucleus staining (blue). GFP‐D4 highly stained the plasma membrane of untreated cells (a), while almost no staining was present on MβCD‐treated cells (b). Analysis by flow cytometry of plasma cholesterol membrane staining with the GFP‐D4 probe. HeLa cells were either left untreated and unstained (HeLa/no probe), untreated (HeLa + GFP‐D4 probe) or treated with MβCD (HeLa/MβCD treatment + GFP‐D4 probe), and next stained; cells were then analyzed by flow cytometry. These representative histograms display the number of cells analyzed (normalized to mode) as a function of GFP‐D4 fluorescence (log intensity). Note that HeLa cells are strongly labeled with the GFP‐D4 probe; MβCD treatment prior to labeling strongly decreases GFP‐D4 signal intensity. Intracellular cholesterol staining with GFP‐D4. HeLa/Ctrl cells were left untreated (a) or treated with U18666A (1 μg/ml; 1 h at 37°C) to promote intracellular cholesterol accumulation (b). After fixation, cells were permeabilized by freezing in liquid nitrogen and incubated with GFP‐D4. Untreated cells (a) were labeled on small discrete structures by the GFP‐D4 probe; in U18666A‐treated cells (b), cholesterol‐filled endosomes were strongly labeled by the GFP‐D4 probe. Whole‐cell cholesterol staining with filipin on fixed HeLa/Ctrl (a) and HeLa/STARD3 (b) cells. Filipin stains cholesterol in the plasma membrane and in intracellular compartments. Note that HeLa/STARD3 cells display intracellular puncta of filipin staining. Intracellular cholesterol staining with filipin. Live HeLa/Ctrl (a) and HeLa/STARD3 (b) cells were treated with MβCD (10 mM in serum‐free medium; 30 min at 37°C) prior to fixation and filipin staining. MβCD treatment removed cholesterol from the plasma membrane and allowed a better visualization of intracellular cholesterol pools. Data information: Higher magnification (3×) images of the area outlined in white are shown on the right. Scale bars: 10 μm.
    Figure Legend Snippet: Cholesterol staining with GFP‐D4 or filipin Plasma membrane cholesterol staining with the GFP‐D4 probe. Live HeLa/Ctrl cells were left untreated (a) or treated with MβCD (b) to remove plasma membrane cholesterol (10 mM in serum‐free medium; 30 min at 37°C), and incubated with GFP‐D4 prior to fixation and nucleus staining (blue). GFP‐D4 highly stained the plasma membrane of untreated cells (a), while almost no staining was present on MβCD‐treated cells (b). Analysis by flow cytometry of plasma cholesterol membrane staining with the GFP‐D4 probe. HeLa cells were either left untreated and unstained (HeLa/no probe), untreated (HeLa + GFP‐D4 probe) or treated with MβCD (HeLa/MβCD treatment + GFP‐D4 probe), and next stained; cells were then analyzed by flow cytometry. These representative histograms display the number of cells analyzed (normalized to mode) as a function of GFP‐D4 fluorescence (log intensity). Note that HeLa cells are strongly labeled with the GFP‐D4 probe; MβCD treatment prior to labeling strongly decreases GFP‐D4 signal intensity. Intracellular cholesterol staining with GFP‐D4. HeLa/Ctrl cells were left untreated (a) or treated with U18666A (1 μg/ml; 1 h at 37°C) to promote intracellular cholesterol accumulation (b). After fixation, cells were permeabilized by freezing in liquid nitrogen and incubated with GFP‐D4. Untreated cells (a) were labeled on small discrete structures by the GFP‐D4 probe; in U18666A‐treated cells (b), cholesterol‐filled endosomes were strongly labeled by the GFP‐D4 probe. Whole‐cell cholesterol staining with filipin on fixed HeLa/Ctrl (a) and HeLa/STARD3 (b) cells. Filipin stains cholesterol in the plasma membrane and in intracellular compartments. Note that HeLa/STARD3 cells display intracellular puncta of filipin staining. Intracellular cholesterol staining with filipin. Live HeLa/Ctrl (a) and HeLa/STARD3 (b) cells were treated with MβCD (10 mM in serum‐free medium; 30 min at 37°C) prior to fixation and filipin staining. MβCD treatment removed cholesterol from the plasma membrane and allowed a better visualization of intracellular cholesterol pools. Data information: Higher magnification (3×) images of the area outlined in white are shown on the right. Scale bars: 10 μm.

    Techniques Used: Staining, Incubation, Flow Cytometry, Cytometry, Fluorescence, Labeling

    59) Product Images from "Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann–Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease"

    Article Title: Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann–Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease

    Journal: Gut

    doi: 10.1136/gutjnl-2015-310382

    Niemann–Pick disease type C1 (NPC1) lysosomal lipid storage disease causes ineffective pathogen clearance despite functional cytokine pathways. (A) Lysosomal lipid storage phenotype in vehicle-treated, U18666A-treated and primary NPC1 monocyte-derived macrophages (MDM). Scale bar, 5 µm. (B) Bacterial killing assay with healthy (n=4–5) and NPC1 patient (n=3–4) MDM and infected with Salmonella typhimurium (left) or adherent-invasive Escherichia coli (AIEC) (right). Individual results are shown as indicated by ticks on x-axes. For each donor, conditions were tested in three parallel infection experiments and colony-forming units (CFU) were normalised to individual CFU without muramyl dipeptide (MDP) stimulation. Red bar represents mean. (C) Cells were assayed as in (B). MDM treated with or without U18666A and MDP pre-stimulation were exposed to S. typhimurium (left, n=4) or AIEC (right, n=4). (D) Representative flow cytometry plots and quantification of tumour necrosis factor (TNF) response in primary NPC1 (n=5) or U18666A-treated (n=4) HLA-DR + CD14 + monocytes following NOD2 or TLR4 stimulation. Mean±SD, grey background indicates normal range calculated from all measured healthy donors. (E) Purification of endogenous ubiquitin (Ub) conjugates using tandem ubiquitin binding entities (TUBE) in lysates of U18666A-treated and control MDM. Purified material and lysate was examined by immunoblotting for indicated proteins. p Values were throughout calculated using Mann–Whitney U test. *p
    Figure Legend Snippet: Niemann–Pick disease type C1 (NPC1) lysosomal lipid storage disease causes ineffective pathogen clearance despite functional cytokine pathways. (A) Lysosomal lipid storage phenotype in vehicle-treated, U18666A-treated and primary NPC1 monocyte-derived macrophages (MDM). Scale bar, 5 µm. (B) Bacterial killing assay with healthy (n=4–5) and NPC1 patient (n=3–4) MDM and infected with Salmonella typhimurium (left) or adherent-invasive Escherichia coli (AIEC) (right). Individual results are shown as indicated by ticks on x-axes. For each donor, conditions were tested in three parallel infection experiments and colony-forming units (CFU) were normalised to individual CFU without muramyl dipeptide (MDP) stimulation. Red bar represents mean. (C) Cells were assayed as in (B). MDM treated with or without U18666A and MDP pre-stimulation were exposed to S. typhimurium (left, n=4) or AIEC (right, n=4). (D) Representative flow cytometry plots and quantification of tumour necrosis factor (TNF) response in primary NPC1 (n=5) or U18666A-treated (n=4) HLA-DR + CD14 + monocytes following NOD2 or TLR4 stimulation. Mean±SD, grey background indicates normal range calculated from all measured healthy donors. (E) Purification of endogenous ubiquitin (Ub) conjugates using tandem ubiquitin binding entities (TUBE) in lysates of U18666A-treated and control MDM. Purified material and lysate was examined by immunoblotting for indicated proteins. p Values were throughout calculated using Mann–Whitney U test. *p

    Techniques Used: Functional Assay, Derivative Assay, Infection, Flow Cytometry, Cytometry, Purification, Binding Assay, MANN-WHITNEY

    Increased bacterial colonisation of Niemann–Pick disease type C1 (NPC1) macrophages is caused by impaired antibacterial autophagy. (A) Flow cytometric analysis of green fluorescent protein (GFP) signal in healthy control monocyte-derived macrophages (MDM) treated with or without U18666A and challenged with GFP- Salmonella typhimurium for 1 h (infection, left) or incubated for one additional hour with gentamicin-containing medium (bacterial clearance, right). External bacteria were quenched with trypan blue. (B) Healthy control MDM were treated with or without U18666A followed by immunoblotting with anti-LC3, anti-p62 and anti-actin. Protein levels of LC3 and p62 were semi-quantified by densitometry relative to actin and expressed as per cent increase to vehicle-treated condition. (C–G) Primary NPC1 mutant and healthy control MDM were infected with GFP- S. typhimurium for 1 h, followed by 1 h culture in gentamicin-containing medium supplemented with Lysotracker (LT) and DAPI staining . Analysis is based on MDM from two patients with NPC1 and four healthy donors. (C) Representative images of infected macrophages. Scale bar, 5 μm. (D) Quantification of total number of bacteria per macrophage. (E) Microscopic assessment of GFP- S. typhimurium according to co-localisation with lysosomes. (F) Percentage of bacteria found in the different stages of autophagic degradation. (G) Absolute numbers of bacteria per MDM for indicated groups. (H) Infection experiment performed as in (C). Additionally, following fixation, MDM were stained with anti-LC3 and co-localisation with bacteria quantified by confocal microscopy. (I) Analysis of LC3-decorated bacteria in primary NPC1 mutant MDM (blue symbol) compared with healthy donor MDM (n=2) or MDM treated with or without U18666A (n=4). Each symbol represents one individual. p Values were determined by Mann–Whitney U test. *p
    Figure Legend Snippet: Increased bacterial colonisation of Niemann–Pick disease type C1 (NPC1) macrophages is caused by impaired antibacterial autophagy. (A) Flow cytometric analysis of green fluorescent protein (GFP) signal in healthy control monocyte-derived macrophages (MDM) treated with or without U18666A and challenged with GFP- Salmonella typhimurium for 1 h (infection, left) or incubated for one additional hour with gentamicin-containing medium (bacterial clearance, right). External bacteria were quenched with trypan blue. (B) Healthy control MDM were treated with or without U18666A followed by immunoblotting with anti-LC3, anti-p62 and anti-actin. Protein levels of LC3 and p62 were semi-quantified by densitometry relative to actin and expressed as per cent increase to vehicle-treated condition. (C–G) Primary NPC1 mutant and healthy control MDM were infected with GFP- S. typhimurium for 1 h, followed by 1 h culture in gentamicin-containing medium supplemented with Lysotracker (LT) and DAPI staining . Analysis is based on MDM from two patients with NPC1 and four healthy donors. (C) Representative images of infected macrophages. Scale bar, 5 μm. (D) Quantification of total number of bacteria per macrophage. (E) Microscopic assessment of GFP- S. typhimurium according to co-localisation with lysosomes. (F) Percentage of bacteria found in the different stages of autophagic degradation. (G) Absolute numbers of bacteria per MDM for indicated groups. (H) Infection experiment performed as in (C). Additionally, following fixation, MDM were stained with anti-LC3 and co-localisation with bacteria quantified by confocal microscopy. (I) Analysis of LC3-decorated bacteria in primary NPC1 mutant MDM (blue symbol) compared with healthy donor MDM (n=2) or MDM treated with or without U18666A (n=4). Each symbol represents one individual. p Values were determined by Mann–Whitney U test. *p

    Techniques Used: Flow Cytometry, Derivative Assay, Infection, Incubation, Mutagenesis, Staining, Confocal Microscopy, MANN-WHITNEY

    Induction of autophagy with chlorpromazine overcomes bacterial killing defect in Niemann–Pick disease type C1 (NPC1). (A) U18666A-treated monocyte-derived macrophages (MDM) were cultured in 0.5–2% of HP-β-cyclodextrin (HPβCD) for last 24 h of U18666A treatment (48 h) and stained with Lysotracker red. Scale bar, 5 μm. (B) Gentamicin protection assay in vehicle- or U18666A-MDM pre-treated with chlorpromazine for 3 h before infection with Salmone lla typhimurium , n=4. Treatment of U18666A-MDM with 2% HPβCD was performed for last 24 h in the presence of U18666A (48 h). Individual results are shown as indicated by ticks on x-axes. For each donor, experimental conditions were tested in three parallel infection experiments and colony-forming units (CFU) were normalised to individual vehicle-treated control. Red bar represents mean. p Values were determined by Mann–Whitney U test comparing results to vehicle-treated control, ***p
    Figure Legend Snippet: Induction of autophagy with chlorpromazine overcomes bacterial killing defect in Niemann–Pick disease type C1 (NPC1). (A) U18666A-treated monocyte-derived macrophages (MDM) were cultured in 0.5–2% of HP-β-cyclodextrin (HPβCD) for last 24 h of U18666A treatment (48 h) and stained with Lysotracker red. Scale bar, 5 μm. (B) Gentamicin protection assay in vehicle- or U18666A-MDM pre-treated with chlorpromazine for 3 h before infection with Salmone lla typhimurium , n=4. Treatment of U18666A-MDM with 2% HPβCD was performed for last 24 h in the presence of U18666A (48 h). Individual results are shown as indicated by ticks on x-axes. For each donor, experimental conditions were tested in three parallel infection experiments and colony-forming units (CFU) were normalised to individual vehicle-treated control. Red bar represents mean. p Values were determined by Mann–Whitney U test comparing results to vehicle-treated control, ***p

    Techniques Used: Derivative Assay, Cell Culture, Staining, Infection, MANN-WHITNEY

    60) 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

    61) Product Images from "Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway"

    Article Title: Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

    Journal: Wellcome open research

    doi: 10.12688/wellcomeopenres.10036.1

    Certain NPC therapeutics promote clearance of intracellular mycobacteria. (A) Cholesterol distribution in wild-type RAW 264.7 macrophages treated with U18666A (2μg/ml) for 48h and subsequently treated with either vehicle (DMSO), curcumin (30μM/24h), miglustat (50μM/72h) or hydroxypropryl-β-cyclodextrin (250μM/24h). Blue, filipin (cholesterol). Scale bar, 5μm. (B) Correlation between extent of infection with mCherry-expressing BCG and levels of RAW 264.7 fluorescence as quantified using flow cytometry (i) Representative histograms showing fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10 or 100 respectively). (ii) Fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10,000 cells counted). Mean ± SEM, N=4. (C) Effect of curcumin on intracellular BCG levels in RAW 264.7 macrophages. Fold change in mean fluorescence of RAW 264.7 macrophages after 48h infection with mCherry-BCG (MOI, 12.5) and subsequent treatment with curcumin (30μM; 24h), miglustat (50μM; 72h), combined curcumin (30μM; 24h) and miglustat (50μM; 72h) or hydroxypropyl-β-cyclodextrin (250μM; 24h). Fold change in fluorescence given relative to untreated, infected control. Mean ± SEM. N=4. * p
    Figure Legend Snippet: Certain NPC therapeutics promote clearance of intracellular mycobacteria. (A) Cholesterol distribution in wild-type RAW 264.7 macrophages treated with U18666A (2μg/ml) for 48h and subsequently treated with either vehicle (DMSO), curcumin (30μM/24h), miglustat (50μM/72h) or hydroxypropryl-β-cyclodextrin (250μM/24h). Blue, filipin (cholesterol). Scale bar, 5μm. (B) Correlation between extent of infection with mCherry-expressing BCG and levels of RAW 264.7 fluorescence as quantified using flow cytometry (i) Representative histograms showing fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10 or 100 respectively). (ii) Fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10,000 cells counted). Mean ± SEM, N=4. (C) Effect of curcumin on intracellular BCG levels in RAW 264.7 macrophages. Fold change in mean fluorescence of RAW 264.7 macrophages after 48h infection with mCherry-BCG (MOI, 12.5) and subsequent treatment with curcumin (30μM; 24h), miglustat (50μM; 72h), combined curcumin (30μM; 24h) and miglustat (50μM; 72h) or hydroxypropyl-β-cyclodextrin (250μM; 24h). Fold change in fluorescence given relative to untreated, infected control. Mean ± SEM. N=4. * p

    Techniques Used: Infection, Expressing, Fluorescence, Flow Cytometry, Cytometry

    62) Product Images from "Trafficking of Endogenous Smooth Muscle Cell Cholesterol: A Role for Serum Amyloid A and Interleukin-1ss"

    Article Title: Trafficking of Endogenous Smooth Muscle Cell Cholesterol: A Role for Serum Amyloid A and Interleukin-1ss

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    doi: 10.1161/ATVBAHA.112.300243

    Serum amyloid A (SAA)–mediated cholesterol trafficking is lysosome–dependent. Smooth muscle cells were treated in the absence or presence of SAA (4 μmol/L) and either no inhibitor (control [Ctl]), chloroquine (Chl; 10 μmol/L), bafilomycin A1 (Baf; 10 nmol/L), or U18666A (U18; 5 μmol/L) with [ 14 C] oleic acid for 24 hours, at which time [ 14 C] cholesteryl oleate accumulation was measured as described in the Materials and Methods section, The data are expressed as [ 14 C] cholesteryl oleate (cpm)/μg protein ± SD (n=3). [ 14 C] cholesteryl oleate accumulation In Chl-treated ( P
    Figure Legend Snippet: Serum amyloid A (SAA)–mediated cholesterol trafficking is lysosome–dependent. Smooth muscle cells were treated in the absence or presence of SAA (4 μmol/L) and either no inhibitor (control [Ctl]), chloroquine (Chl; 10 μmol/L), bafilomycin A1 (Baf; 10 nmol/L), or U18666A (U18; 5 μmol/L) with [ 14 C] oleic acid for 24 hours, at which time [ 14 C] cholesteryl oleate accumulation was measured as described in the Materials and Methods section, The data are expressed as [ 14 C] cholesteryl oleate (cpm)/μg protein ± SD (n=3). [ 14 C] cholesteryl oleate accumulation In Chl-treated ( P

    Techniques Used: CTL Assay

    63) 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

    64) Product Images from "Camphene, a Plant Derived Monoterpene, Exerts Its Hypolipidemic Action by Affecting SREBP-1 and MTP Expression"

    Article Title: Camphene, a Plant Derived Monoterpene, Exerts Its Hypolipidemic Action by Affecting SREBP-1 and MTP Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147117

    Effect of camphene on lipid profile and metabolism in HepG2 cells-Comparison with well known hypolipidemic compounds. On day 7, the cells were incubated with the compounds for 24 h in DMEM containing 10% (v/v) human LPDS. HepG2 cells were treated with camphene (lanes: 2–4), mevinolin (lane: 5), atorvastatin (lane: 7), the OSC inhibitor, U18666A (lane: 6) and the ACAT inhibitor, F1394 (lane: 8). Control-non treated cells migrate in places 1 and 9. Camphene was used at concentration of 25, 50 and 100 μM lanes 2, 3 and 4 respectively. Four hours prior to the end of the incubation period, the cells were pulse-labeled with [ 14 C]-acetic acid, sodium salt. This panel shows the autoradiograph of synthesized intracellular lipids separated by TLC after labelling of cells with [ 14 C]-acetate. The positions of the different lipids have been determined using non radioactive standards. The arrows indicate the positions of migration of cholesterol, fatty acids, triglycerides, cholesterol esters, MOS and DOS.
    Figure Legend Snippet: Effect of camphene on lipid profile and metabolism in HepG2 cells-Comparison with well known hypolipidemic compounds. On day 7, the cells were incubated with the compounds for 24 h in DMEM containing 10% (v/v) human LPDS. HepG2 cells were treated with camphene (lanes: 2–4), mevinolin (lane: 5), atorvastatin (lane: 7), the OSC inhibitor, U18666A (lane: 6) and the ACAT inhibitor, F1394 (lane: 8). Control-non treated cells migrate in places 1 and 9. Camphene was used at concentration of 25, 50 and 100 μM lanes 2, 3 and 4 respectively. Four hours prior to the end of the incubation period, the cells were pulse-labeled with [ 14 C]-acetic acid, sodium salt. This panel shows the autoradiograph of synthesized intracellular lipids separated by TLC after labelling of cells with [ 14 C]-acetate. The positions of the different lipids have been determined using non radioactive standards. The arrows indicate the positions of migration of cholesterol, fatty acids, triglycerides, cholesterol esters, MOS and DOS.

    Techniques Used: Incubation, Concentration Assay, Labeling, Autoradiography, Synthesized, Thin Layer Chromatography, Migration

    65) Product Images from "Niemann-Pick C1 Affects the Gene Delivery Efficacy of Degradable Polymeric Nanoparticles"

    Article Title: Niemann-Pick C1 Affects the Gene Delivery Efficacy of Degradable Polymeric Nanoparticles

    Journal: ACS Nano

    doi: 10.1021/nn501630h

    U18666A inhibits C32–122-mediated uptake of DNA in MEFs. (a) MEFs were transfected with C32–122 polyplexes containing Cy3-labeled DNA in the presence of various endocytic pathway inhibitors (5 μM U18666A, 50 μM dynasore, 1 mM methyl-β-cyclodextrin, 10 μM genistein, 5 μM filipin, 5 μM chlorpromazine, 10 μM EIPA). MEFs were pretreated with the inhibitors 1 h prior to transfection. After 3 h, the cells were washed, fixed, treated with a nuclear stain, and analyzed by high-throughput confocal microscopy to quantify relative DNA uptake (mean ± SD, n = 3); * indicates p
    Figure Legend Snippet: U18666A inhibits C32–122-mediated uptake of DNA in MEFs. (a) MEFs were transfected with C32–122 polyplexes containing Cy3-labeled DNA in the presence of various endocytic pathway inhibitors (5 μM U18666A, 50 μM dynasore, 1 mM methyl-β-cyclodextrin, 10 μM genistein, 5 μM filipin, 5 μM chlorpromazine, 10 μM EIPA). MEFs were pretreated with the inhibitors 1 h prior to transfection. After 3 h, the cells were washed, fixed, treated with a nuclear stain, and analyzed by high-throughput confocal microscopy to quantify relative DNA uptake (mean ± SD, n = 3); * indicates p

    Techniques Used: Transfection, Labeling, Staining, High Throughput Screening Assay, Confocal Microscopy

    66) Product Images from "Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes"

    Article Title: Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200110138

    Expression of PLP in BHK cells affects the sorting of GPI–YFP. (a) BHK cells were transfected with GPI–YFP and, after 24 h, were fixed and analyzed by immunofluorescence. (b) Cells were transfected with GPI–YFP, incubated for 24 h in the presence of 10 μg/ml U18666A, fixed, and analyzed by immunofluorescence. (c) BHK cells were cotransfected with PLP and GPI–YFP and analyzed after 24 h by immunofluorescence for PLP (red) and GPI–YFP (green). In the merged image, yellow indicates colocalization of PLP and GPI–YFP. Bars, 10 μm.
    Figure Legend Snippet: Expression of PLP in BHK cells affects the sorting of GPI–YFP. (a) BHK cells were transfected with GPI–YFP and, after 24 h, were fixed and analyzed by immunofluorescence. (b) Cells were transfected with GPI–YFP, incubated for 24 h in the presence of 10 μg/ml U18666A, fixed, and analyzed by immunofluorescence. (c) BHK cells were cotransfected with PLP and GPI–YFP and analyzed after 24 h by immunofluorescence for PLP (red) and GPI–YFP (green). In the merged image, yellow indicates colocalization of PLP and GPI–YFP. Bars, 10 μm.

    Techniques Used: Expressing, Plasmid Purification, Transfection, Immunofluorescence, Incubation

    67) 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

    68) Product Images from "The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution"

    Article Title: The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709123

    RELCH, OSBP, and Rab11 depletion results in less cholesterol accumulation in the TGN and ER. (A and B) The homogenates from the shRNA-expressing HeLa cells were fractionated using a Histodenz step density gradient. (A) The fractions were immunoblotted (IB) with antibodies against TGN46, calnexin, and Lamp2. (B) Percentages of cholesterol (µg/mg protein) in the TGN (fractions 4–6), LEs/lysosomes (LE/LY; 7–9), or ER (10 and 11) in the total fractions are shown in the bar graph. (C) HeLa cells were treated with 2 µg/ml U18666A for 16 h and stained with Filipin. Bar, 20 µm. (D–G) Immunoisolation of ER, TGN, and LE/lysosome from the PNS derived from the U18666A-treated (D and E) or RELCH-, Rab11a/b-, and OSBP-depleted cells by shRNAs (F and G). ER, TGN, and LE/lysosome membranes were isolated using calnexin, TGN46, and Lamp1 antibodies, respectively. (D and F) The isolated samples were immunoblotted with calnexin, TGN46, and Lamp2 antibodies. (E and G) Quantification of the cholesterol content in the isolated membranes (relative to the control samples). Data are expressed as means ± SEM from at least three independent experiments. *, P
    Figure Legend Snippet: RELCH, OSBP, and Rab11 depletion results in less cholesterol accumulation in the TGN and ER. (A and B) The homogenates from the shRNA-expressing HeLa cells were fractionated using a Histodenz step density gradient. (A) The fractions were immunoblotted (IB) with antibodies against TGN46, calnexin, and Lamp2. (B) Percentages of cholesterol (µg/mg protein) in the TGN (fractions 4–6), LEs/lysosomes (LE/LY; 7–9), or ER (10 and 11) in the total fractions are shown in the bar graph. (C) HeLa cells were treated with 2 µg/ml U18666A for 16 h and stained with Filipin. Bar, 20 µm. (D–G) Immunoisolation of ER, TGN, and LE/lysosome from the PNS derived from the U18666A-treated (D and E) or RELCH-, Rab11a/b-, and OSBP-depleted cells by shRNAs (F and G). ER, TGN, and LE/lysosome membranes were isolated using calnexin, TGN46, and Lamp1 antibodies, respectively. (D and F) The isolated samples were immunoblotted with calnexin, TGN46, and Lamp2 antibodies. (E and G) Quantification of the cholesterol content in the isolated membranes (relative to the control samples). Data are expressed as means ± SEM from at least three independent experiments. *, P

    Techniques Used: shRNA, Expressing, Staining, Derivative Assay, Isolation

    69) 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

    70) Product Images from "Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts"

    Article Title: Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165004

    U18666A modifies the distribution of mature HYAL1 in a sucrose gradient, similarly to lysosomal markers. An M+L+P fraction was prepared from control or U18666A treated osteoclasts and centrifuged in a linear sucrose density gradient extending from 1.04 g/mL to 1.26 g/mL. Nine fractions were collected from top to bottom. (A-B) The activities of the markers β-galactosidase (A) and alkaline α-glucosidase (B) were measured using fluorometric assay in the different fractions. The ordinate of the graphs corresponds to the frequency (Q/SQ.r, where Q represents the activity found in the fraction, SQ, the total activity recovered in the sum of the fractions, and r, the increment of density from top to bottom of the fraction). (C-F) Cathepsin K and HYAL1 were detected by western blotting (reducing conditions) in the fractions collected after centrifugation. The precursor and mature forms of these proteins are shown by closed and open arrowheads, respectively. Of note, a longer exposure time is shown for the upper part of the blots shown in panels C and D, to visualize cathepsin K proforms.
    Figure Legend Snippet: U18666A modifies the distribution of mature HYAL1 in a sucrose gradient, similarly to lysosomal markers. An M+L+P fraction was prepared from control or U18666A treated osteoclasts and centrifuged in a linear sucrose density gradient extending from 1.04 g/mL to 1.26 g/mL. Nine fractions were collected from top to bottom. (A-B) The activities of the markers β-galactosidase (A) and alkaline α-glucosidase (B) were measured using fluorometric assay in the different fractions. The ordinate of the graphs corresponds to the frequency (Q/SQ.r, where Q represents the activity found in the fraction, SQ, the total activity recovered in the sum of the fractions, and r, the increment of density from top to bottom of the fraction). (C-F) Cathepsin K and HYAL1 were detected by western blotting (reducing conditions) in the fractions collected after centrifugation. The precursor and mature forms of these proteins are shown by closed and open arrowheads, respectively. Of note, a longer exposure time is shown for the upper part of the blots shown in panels C and D, to visualize cathepsin K proforms.

    Techniques Used: Activity Assay, Western Blot, Centrifugation

    71) Product Images from "TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes"

    Article Title: TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01871-z

    TFEB/3 and SREBF2 co-operate to regulate TMEM55B levels in response to changes in cholesterol levels. a , b HeLa cells were treated with drugs to deplete cellular cholesterol for 120 h, treated with 10 μM U18666A for 18 h, or left untreated. a Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH mRNA, using two-tailed t-test ** P
    Figure Legend Snippet: TFEB/3 and SREBF2 co-operate to regulate TMEM55B levels in response to changes in cholesterol levels. a , b HeLa cells were treated with drugs to deplete cellular cholesterol for 120 h, treated with 10 μM U18666A for 18 h, or left untreated. a Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH mRNA, using two-tailed t-test ** P

    Techniques Used: Real-time Polymerase Chain Reaction, Two Tailed Test

    72) Product Images from "Identification of an Intracellular Site of Prion Conversion"

    Article Title: Identification of an Intracellular Site of Prion Conversion

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000426

    U18666A blocks transferrin trafficking from EE to ERC but does not interfere with Tfn delivery to the cell surface. (A) Recycling of Alexa 488-transferrin (Tfn) was analyzed in control ScGT1 cells. A diffused cholesterol distribution was revealed by filipin staining (blue). Yellow color and arrowheads indicate colocalization between Tfn and EEA-1. Inset represents magnification of the boxed area. (B) The same experiment described in (A) was performed in ScGT1 cells treated with U18666A for 6 days. Characteristic large cholesterol laden endosomes were revealed by fillipin staining (blue). Note that in contrast to control cells, in treated cells Tfn was not transported to ERC 15 min post-internalization. Alexa 488-transferrin was chased out of both control (C) and U18666A (D) treated cells after 45 min. Scale bars represent 10 µm.
    Figure Legend Snippet: U18666A blocks transferrin trafficking from EE to ERC but does not interfere with Tfn delivery to the cell surface. (A) Recycling of Alexa 488-transferrin (Tfn) was analyzed in control ScGT1 cells. A diffused cholesterol distribution was revealed by filipin staining (blue). Yellow color and arrowheads indicate colocalization between Tfn and EEA-1. Inset represents magnification of the boxed area. (B) The same experiment described in (A) was performed in ScGT1 cells treated with U18666A for 6 days. Characteristic large cholesterol laden endosomes were revealed by fillipin staining (blue). Note that in contrast to control cells, in treated cells Tfn was not transported to ERC 15 min post-internalization. Alexa 488-transferrin was chased out of both control (C) and U18666A (D) treated cells after 45 min. Scale bars represent 10 µm.

    Techniques Used: Staining

    U18666A reduces PrP Sc levels and impairs PrP C trafficking in ScGT1 cells, without affecting PrP distribution in DRMs. (A) Levels of total PrP (PK−) or PrP Sc (PK+) were analyzed on western blot in control cells (ctrl) and cells treated with U18666A. Increased PrP C and decreased PrP Sc levels were observed in treated cells. (B) Lysates from control and U18666A treated ScGT1 cells were applied on sucrose gradients and ultracentrifuged at 200000 g for 16 hr. Twelve fractions were collected and proteins were precipitated using 10% of trichloroacetic acid. PrP and flotillin-1 contents in each fraction were analyzed on western blot using SAF61 mAb and flotillin-1 mAb. Fractions 4–7 correspond to detergent resistant membranes (DRM) based on distribution of flotillin-1, which is mainly present in DRMs. In ScGT1 cells PrP is distributed in DRMs and U18666A treatment does not change its distribution. (C) Steady-state localization of PrP C was analyzed in control (ctrl) and U18666A-treated ScGT1 cells. Yellow color and arrowheads represent colocalization between PrP C and EEA-1. Scale bars represent 10 µm (D) Quantification results are presented as % (mean±s.e.m, n = 80) of total PrP C signal (red) colocalizing with EEA-1 signal (green) (p = 0,014, t-test).
    Figure Legend Snippet: U18666A reduces PrP Sc levels and impairs PrP C trafficking in ScGT1 cells, without affecting PrP distribution in DRMs. (A) Levels of total PrP (PK−) or PrP Sc (PK+) were analyzed on western blot in control cells (ctrl) and cells treated with U18666A. Increased PrP C and decreased PrP Sc levels were observed in treated cells. (B) Lysates from control and U18666A treated ScGT1 cells were applied on sucrose gradients and ultracentrifuged at 200000 g for 16 hr. Twelve fractions were collected and proteins were precipitated using 10% of trichloroacetic acid. PrP and flotillin-1 contents in each fraction were analyzed on western blot using SAF61 mAb and flotillin-1 mAb. Fractions 4–7 correspond to detergent resistant membranes (DRM) based on distribution of flotillin-1, which is mainly present in DRMs. In ScGT1 cells PrP is distributed in DRMs and U18666A treatment does not change its distribution. (C) Steady-state localization of PrP C was analyzed in control (ctrl) and U18666A-treated ScGT1 cells. Yellow color and arrowheads represent colocalization between PrP C and EEA-1. Scale bars represent 10 µm (D) Quantification results are presented as % (mean±s.e.m, n = 80) of total PrP C signal (red) colocalizing with EEA-1 signal (green) (p = 0,014, t-test).

    Techniques Used: Western Blot

    73) 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

    74) 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

    75) Product Images from "Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking"

    Article Title: Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2009.07.003

    Loss of NgBR induces free cholesterol accumulation in cells (A) EA.hy926 cells or (B) NgBR +/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p
    Figure Legend Snippet: Loss of NgBR induces free cholesterol accumulation in cells (A) EA.hy926 cells or (B) NgBR +/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p

    Techniques Used: Incubation, Small Interfering RNA, Positive Control, Inhibition, Staining, Infection, Construct, Expressing, Transfection

    Related Articles

    Immunostaining:

    Article Title: Histone Deacetylase Inhibition Decreases Cholesterol Levels in Neuronal Cells by Modulating Key Genes in Cholesterol Synthesis, Uptake and Efflux
    Article Snippet: Reagents and antibodies The chemical inhibitors TSA and U18666A {3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one} were from Sigma (Sigma Aldrich Inc., St Louis, MO, USA). .. The secondary antibodies anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 488 (Molecular Probes®, Invitrogen, Carlsbad, CA, USA) were used in the immunostaining.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies
    Article Snippet: For quantification of HBsAg, an Enzygnost HBsAg enzyme-linked immunosorbent assay (ELISA; Siemens, Germany) was used. .. U18666A, which inhibits the transportation of cholesterol ( ) and alters the trafficking of MVB-associated membrane proteins ( , ), was ordered from Sigma-Aldrich (St. Louis, MO).

    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. ..

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    Article Snippet: .. At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above. ..

    Cell Culture:

    Article Title: Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease
    Article Snippet: Paragraph title: Cell culture ... 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.

    Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action
    Article Snippet: Paragraph title: Cell Culture ... Cells were treated for specified periods of time with 0.1% DMSO as a vehicle control or 10 µg/ml U18666a (Sigma-Aldrich).

    Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages
    Article Snippet: Cells and reagents HeLa cells were cultured in Dulbecco modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen, Cergy-Pontoise, France), 2 mM glutamine, 100 UI/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich, St Louis, MO). .. U18666A (Sigma-Aldrich) was used at 3 µg/ml in 1% LPDS medium.

    Expressing:

    Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action
    Article Snippet: HA-GLUT4 expressing 3T3-L1 adipocytes were generated as previously described . .. Cells were treated for specified periods of time with 0.1% DMSO as a vehicle control or 10 µg/ml U18666a (Sigma-Aldrich).

    Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages
    Article Snippet: U18666A (Sigma-Aldrich) was used at 3 µg/ml in 1% LPDS medium. .. HeLa cells expressing a GFP tagged M6PR were described in .

    Modification:

    Article Title: Gpnmb Is a Potential Marker for the Visceral Pathology in Niemann-Pick Type C Disease
    Article Snippet: Cell culture RAW264.7 cells were obtained from the American Type Culture Collection and were cultured in DMEM (Dulbecco’s Modified Eagle Medium)/10% FCS (fetal calf serum) supplemented with penicillin/streptomycin. .. 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.

    Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action
    Article Snippet: Cell Culture 3T3-L1 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% foetal calf serum (Thermo Scientific) and 1% Glutamax (Life Technologies) at 37°C in 10% CO2 . .. Cells were treated for specified periods of time with 0.1% DMSO as a vehicle control or 10 µg/ml U18666a (Sigma-Aldrich).

    Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages
    Article Snippet: Cells and reagents HeLa cells were cultured in Dulbecco modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen, Cergy-Pontoise, France), 2 mM glutamine, 100 UI/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich, St Louis, MO). .. U18666A (Sigma-Aldrich) was used at 3 µg/ml in 1% LPDS medium.

    Double Immunofluorescence Staining:

    Article Title: Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies
    Article Snippet: For double immunofluorescence staining, a donkey-derived mouse-specific Alexa 488-labeled secondary antibody from Invitrogen (Darmstadt, Germany) and a donkey-derived rabbit-specific Cy3-labeled secondary antibody were used (Jackson Laboratories, Bar Harbor, ME). .. U18666A, which inhibits the transportation of cholesterol ( ) and alters the trafficking of MVB-associated membrane proteins ( , ), was ordered from Sigma-Aldrich (St. Louis, MO).

    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. ..

    Concentration Assay:

    Article Title: The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus
    Article Snippet: .. U18666A (Sigma-Aldrich) was used at a concentration of 2 μg/ml for 16 h if not indicated differently. .. Simvastatin (Selleckchem) was used at a concentration of 10 μM.

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    Article Snippet: .. At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above. ..

    Protease Inhibitor:

    Article Title: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection
    Article Snippet: Materials We obtained U18666A, Dulbecco’s phosphate-buffered saline (PBS), CuSO4 , Tris-HCl, NaCl, FCS, chloroquine, biotin, thiourea, and Benzonase Nuclease from Sigma-Aldrich, St. Louis, MO; [1-14 C]oleic acid (55 mCi/mmol) from Perkin Elmer, Waltham, MA; Zeocin and pcDNA3.1/Zeo(-) from Life Technologies, Grand Island, NY; and FuGENE HD from Promega, Madison, WI. .. Protein A/G Plus-Agarose Immunoprecipitation Reagent:sc 2003 from Santa Cruz Biotechnology, Dallas, TX; cOmplete, EDTA-free Protease Inhibitor Cocktail and Nonidet P40 (NP-40) from Roche Diagnostics, Indianapolis, IN; Alexa Fluor 532 Azide from Joseph M. Ready (U.T.

    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 . ..

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    Article Snippet: HeLa cells (5 × 104 ) were infected with mCherry-C. burnetii in 6-well plates for 1 h. At 2 days postinfection, the cells were trypsinized and resuspended to 1 × 105 cells/ml, and plated onto ibidi-treated channel µslide VI0.4 (3 × 103 cells per channel; Ibidi). .. At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above.

    Generated:

    Article Title: Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content
    Article Snippet: LAMP-1−/− , LAMP-2−/− , LAMPnull and wt mouse embryonic fibroblasts (MEFs) were generated as previously described . .. Cells were pre-treated with U18666A (0.25-3 μg/ml), quinacrine (2 μM) and 25-HC (1 μg/ml; all from Sigma-Aldrich, St. Louis, MO, USA) for 48 h. MβCD (400–500 μM; Sigma-Aldrich) was added to cells for 1 h to allow endocytosis and then removed and cells were chased for 24 h. This approach was shown to deplete cholesterol from the lysosomal membrane rather than the plasma membrane .

    Article Title: Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis
    Article Snippet: Differentiation of progenitor cells The generated neural progenitor cells, differentiated from patient-specific iPS cells [ ], were used for 20 passages and seeded at an expansion density of 100,000 cells/cm2 on poly-L-ornithine-coated (15 μg/ml; Sigma, Germany)/laminin (10 μg/ml; Trevigen, USA) dishes in proliferation medium containing DMEM, 30% DMEM/F-12, 1X B27, 0.5% penicillin/streptomycin, 20 ng/ml FGF2 (Amsbio, United Kingdom), 20 ng/ml EGF (Peprotech, Germany). .. For experiments comprising an application of PMA or U18666A, cells were differentiated for 40 days and PMA (10 nM, Cayman Chemicals, USA) or U18666A (1 μg/ml Sigma, Germany) were applied for 24 h or 48 h.

    Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action
    Article Snippet: HA-GLUT4 expressing 3T3-L1 adipocytes were generated as previously described . .. Cells were treated for specified periods of time with 0.1% DMSO as a vehicle control or 10 µg/ml U18666a (Sigma-Aldrich).

    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).

    Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *
    Article Snippet: Cholesterol trafficking was arrested by treating MA-10 cells with 7 μ m U18666A (Sigma) for 4 h and re-induced by treating them with Bt2 cAMP.

    Fluorescence:

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    Article Snippet: PVs with visible mCherry fluorescence in the PV lumen were scored as “lytic PVs” with 50 PVs scored for each condition for three individual experiments. .. At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above.

    Isolation:

    Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages
    Article Snippet: U18666A (Sigma-Aldrich) was used at 3 µg/ml in 1% LPDS medium. .. Monocytes were isolated from human blood of normolipidemic donors by ficoll density gradient (Eurobio, les Ulis, France) and subsequently differentiated into human monocytes-derived macrophages (HMDM) by adhesion on plastic Primaria plates (Falcon BD, Franklin Lakes, NJ,) for 7 days in RPM1640 medium, supplemented with 10% heat-inactived FCS, 2 mM glutamine, 100 UI/ml penicillin, 100 µg/ml streptomycin, and 20 ng/ml hM-CSF (human macrophages colony-stimulating factor) (AbCys, Paris, France).

    Microscopy:

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    Article Snippet: The medium was changed daily, and cells were examined live every 24 h on a Leica inverted DMI6000B microscope with a 63× oil immersion objective. .. At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above.

    Purification:

    Article Title: Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies
    Article Snippet: Purified mouse anti-GM130 clone 35 as a marker for the Golgi apparatus and protein disulfide isomerase (PDI)-specific antibody were ordered from BD Transduction Laboratories. .. U18666A, which inhibits the transportation of cholesterol ( ) and alters the trafficking of MVB-associated membrane proteins ( , ), was ordered from Sigma-Aldrich (St. Louis, MO).

    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).

    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.

    Quantitation Assay:

    Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    Article Snippet: Paragraph title: Quantitation of lytic PVs containing lysed C. burnetii. ... At 3 days postinfection, dimethyl sulfoxide (DMSO) control, U18666A (1 or 5 µM), or the indicated FDA-approved drugs (see in the supplemental material; obtained from Sigma and used at a final concentration of 20 µM) with or without vATPase inhibitor bafilomycin A1 (100 nM) were added to the cells and incubated for the time indicated prior to counting lytic PVs as described above.

    Knock-Out:

    Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action
    Article Snippet: Cells were treated for specified periods of time with 0.1% DMSO as a vehicle control or 10 µg/ml U18666a (Sigma-Aldrich). .. Control (K1) and NPC1 knockout (2–2) CHO cells were a kind gift from Dr. Andrew Brown (UNSW, Sydney, Australia).

    Immunoprecipitation:

    Article Title: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection
    Article Snippet: Materials We obtained U18666A, Dulbecco’s phosphate-buffered saline (PBS), CuSO4 , Tris-HCl, NaCl, FCS, chloroquine, biotin, thiourea, and Benzonase Nuclease from Sigma-Aldrich, St. Louis, MO; [1-14 C]oleic acid (55 mCi/mmol) from Perkin Elmer, Waltham, MA; Zeocin and pcDNA3.1/Zeo(-) from Life Technologies, Grand Island, NY; and FuGENE HD from Promega, Madison, WI. .. Protein A/G Plus-Agarose Immunoprecipitation Reagent:sc 2003 from Santa Cruz Biotechnology, Dallas, TX; cOmplete, EDTA-free Protease Inhibitor Cocktail and Nonidet P40 (NP-40) from Roche Diagnostics, Indianapolis, IN; Alexa Fluor 532 Azide from Joseph M. Ready (U.T.

    Marker:

    Article Title: Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies
    Article Snippet: Purified mouse anti-GM130 clone 35 as a marker for the Golgi apparatus and protein disulfide isomerase (PDI)-specific antibody were ordered from BD Transduction Laboratories. .. U18666A, which inhibits the transportation of cholesterol ( ) and alters the trafficking of MVB-associated membrane proteins ( , ), was ordered from Sigma-Aldrich (St. Louis, MO).

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    Millipore u18666a
    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 <t>U18666a</t> (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.
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    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

    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

    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