filipin iii  (Millipore)


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    Name:
    Filipin III ready made solution
    Description:
    Filipin III is a polyene macrolide antibiotic and the major component isolated from cultures of S filipinensis 1 Due to its ability to fluoresce and bind to cholesterol Filipin has been widelyused as a probe for sterol location in biological membranes 2 and has been used clinically as a stain for free cholesterol in the study of Type C Niemann Pick disease 3 Filipin inhibits prion protein PrP endocytosis and causes the release of PrP from the plasma membrane 4 Filipin III was found to trigger signaling responses in tobacco cells including a NADPH oxidase dependent production 5 The antifungal mechanism of action may be due to altering membrane permeability and associated functions by binding to membrane sterols Filipin inhibits prion protein PrP endocytosis and causes the release of PrP from the plasma membrane
    Catalog Number:
    sae0087
    Price:
    None
    Applications:
    Filipin has been used in a double staining procedure as a probe for the detection of lipoproteins in polyacrylamide gel and immobilized on nitrocellulose membranes. It is also widely used to localize and quantitate unesterified cholesterol by virtue of a specific fluorescent complex.
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    Structured Review

    Millipore filipin iii
    Filipin III ready made solution
    Filipin III is a polyene macrolide antibiotic and the major component isolated from cultures of S filipinensis 1 Due to its ability to fluoresce and bind to cholesterol Filipin has been widelyused as a probe for sterol location in biological membranes 2 and has been used clinically as a stain for free cholesterol in the study of Type C Niemann Pick disease 3 Filipin inhibits prion protein PrP endocytosis and causes the release of PrP from the plasma membrane 4 Filipin III was found to trigger signaling responses in tobacco cells including a NADPH oxidase dependent production 5 The antifungal mechanism of action may be due to altering membrane permeability and associated functions by binding to membrane sterols Filipin inhibits prion protein PrP endocytosis and causes the release of PrP from the plasma membrane
    https://www.bioz.com/result/filipin iii/product/Millipore
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    filipin iii - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding * "

    Article Title: S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807009200

    Effect of OA and filipin III on binding of FITC-SNO-HSA to HepG2 cells. To prepare FITC-SNO-HSA, HSA was first S -nitrosylated as described above, followed by FITC labeling. FITC-SNO-HSA (50 μg/ml) with varying OA content (0, 3, 5 OA/HSA) was dissolved in 10 m m phosphate-buffered saline (pH 7.4) and added to HepG2 cells (5 × 10 5 cells/well) for 10 min at 4 °C. In some experiments, the cells had been pretreated with 50 μ m filipin III for 30 min. After incubation, the cells were washed twice with the phosphate-buffered saline to remove unbound FITC-SNO-HSA. After washing, the cells were analyzed using a fluorescence microscope. E , four columns quantify the FITC-fluorescence in panels A, B, C , and D , respectively, using the fluorescence in panel A as a reference value, i.e. 100%. The values are means ± S.E. ( n = 3). * , p
    Figure Legend Snippet: Effect of OA and filipin III on binding of FITC-SNO-HSA to HepG2 cells. To prepare FITC-SNO-HSA, HSA was first S -nitrosylated as described above, followed by FITC labeling. FITC-SNO-HSA (50 μg/ml) with varying OA content (0, 3, 5 OA/HSA) was dissolved in 10 m m phosphate-buffered saline (pH 7.4) and added to HepG2 cells (5 × 10 5 cells/well) for 10 min at 4 °C. In some experiments, the cells had been pretreated with 50 μ m filipin III for 30 min. After incubation, the cells were washed twice with the phosphate-buffered saline to remove unbound FITC-SNO-HSA. After washing, the cells were analyzed using a fluorescence microscope. E , four columns quantify the FITC-fluorescence in panels A, B, C , and D , respectively, using the fluorescence in panel A as a reference value, i.e. 100%. The values are means ± S.E. ( n = 3). * , p

    Techniques Used: Binding Assay, Labeling, Incubation, Fluorescence, Microscopy

    Intracellular NO concentration of HepG2 cells exposed to SNO-HSA with different molar ratios of OA. The HepG2 cells (5 × 10 5 cells/well) were first incubated with 5 μ m DAF-FM DA for 1 h and then treated with 100 μ m SNO-HSA with different amounts of bound OA in 10 m m phosphate-buffered saline, pH 7.4 in the dark at 37 °C. Some of the experiments with the highest OA concentration were also performed in the presence of 50 μ m filipin III. Intracellular NO was monitored with DAF-FM fluorescence (ex/em of 385/535 nm). Δ fluorescence represents DAF-FM fluorescence in cells incubated with different preparations of SNO-HSA, minus the fluorescence in cells that had been incubated with buffer only. Data are expressed as means ± S.E. ( n = 4); the bars showing S.E. were smaller than the size of the symbols.
    Figure Legend Snippet: Intracellular NO concentration of HepG2 cells exposed to SNO-HSA with different molar ratios of OA. The HepG2 cells (5 × 10 5 cells/well) were first incubated with 5 μ m DAF-FM DA for 1 h and then treated with 100 μ m SNO-HSA with different amounts of bound OA in 10 m m phosphate-buffered saline, pH 7.4 in the dark at 37 °C. Some of the experiments with the highest OA concentration were also performed in the presence of 50 μ m filipin III. Intracellular NO was monitored with DAF-FM fluorescence (ex/em of 385/535 nm). Δ fluorescence represents DAF-FM fluorescence in cells incubated with different preparations of SNO-HSA, minus the fluorescence in cells that had been incubated with buffer only. Data are expressed as means ± S.E. ( n = 4); the bars showing S.E. were smaller than the size of the symbols.

    Techniques Used: Concentration Assay, Incubation, Fluorescence

    2) Product Images from "Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse"

    Article Title: Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00779.2011

    Cholesterol localization at the cone synapse and effects of pharmacological treatments on retinal cholesterol levels. A : to visualize membrane cholesterol, an enzymatically dissociated double cone was fixed and stained with filipin III (300 μg/ml
    Figure Legend Snippet: Cholesterol localization at the cone synapse and effects of pharmacological treatments on retinal cholesterol levels. A : to visualize membrane cholesterol, an enzymatically dissociated double cone was fixed and stained with filipin III (300 μg/ml

    Techniques Used: Staining

    3) Product Images from "Phosphate effect on filipin production and morphological differentiation in Streptomyces filipinensis and the role of the PhoP transcription factor"

    Article Title: Phosphate effect on filipin production and morphological differentiation in Streptomyces filipinensis and the role of the PhoP transcription factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208278

    Phosphate consumption in YEME medium by S . filipinensis . Fermentations were carried out at 30°C in YEME medium supplemented with variable concentrations of inorganic phosphate (0, 1, 2.5 and 10 mM). The consumption of inorganic phosphate (broken lines) and the specific production of filipin (solid line) were determined. The vertical bars represent the standard deviation between three biological replicates.
    Figure Legend Snippet: Phosphate consumption in YEME medium by S . filipinensis . Fermentations were carried out at 30°C in YEME medium supplemented with variable concentrations of inorganic phosphate (0, 1, 2.5 and 10 mM). The consumption of inorganic phosphate (broken lines) and the specific production of filipin (solid line) were determined. The vertical bars represent the standard deviation between three biological replicates.

    Techniques Used: Standard Deviation

    Phosphate effect on filipin III production. A) Effect of increasing inorganic phosphate concentrations (0, 1, 5, and 10 mM) on the specific production of filipin (expressed as μg of filipin per mg of dry weight) by the wild type S . filipinensis DSM 40112 in YEME medium. B) Determination of the threshold for saturation of phosphate depressive effect on filipin biosynthesis. Volumetric production of filipin is indicated in orange and growth in green. Data are the average of three duplicate flasks. Vertical bars indicate standard deviation of the mean values.
    Figure Legend Snippet: Phosphate effect on filipin III production. A) Effect of increasing inorganic phosphate concentrations (0, 1, 5, and 10 mM) on the specific production of filipin (expressed as μg of filipin per mg of dry weight) by the wild type S . filipinensis DSM 40112 in YEME medium. B) Determination of the threshold for saturation of phosphate depressive effect on filipin biosynthesis. Volumetric production of filipin is indicated in orange and growth in green. Data are the average of three duplicate flasks. Vertical bars indicate standard deviation of the mean values.

    Techniques Used: Standard Deviation

    Both PhoP or PhoRP inactivation increase filipin production and gene complementation restores it. A) Time course quantification of filipin III production and growth curves in the wild-type and mutant strains. Fermentations were carried out at 30°C in YEME medium supplemented with variable concentrations of inorganic phosphate (0, 1 and 2.5 mM). B and C) Effects of gene complementation in YEME medium in the presence of 1 mM supplemented phosphate. Data are the average of three duplicate flasks. Vertical bars indicate standard deviation of the mean values.
    Figure Legend Snippet: Both PhoP or PhoRP inactivation increase filipin production and gene complementation restores it. A) Time course quantification of filipin III production and growth curves in the wild-type and mutant strains. Fermentations were carried out at 30°C in YEME medium supplemented with variable concentrations of inorganic phosphate (0, 1 and 2.5 mM). B and C) Effects of gene complementation in YEME medium in the presence of 1 mM supplemented phosphate. Data are the average of three duplicate flasks. Vertical bars indicate standard deviation of the mean values.

    Techniques Used: Mutagenesis, Standard Deviation

    4) Product Images from "Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1"

    Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1056-1

    LAMP-1 is essential for cyclodextrin-mediated cholesterol export from lysosomes. Verification of HeLa cells either overexpressing LAMP-1 (HeLa-LE) or stably carrying shRNA for LAMP-1 (Hela LAMP-1 knockdown or HeLa-kd) by western blot ( a ). HeLa-LE shows significantly increased levels of LAMP-1, while HeLa-Lkd shows decreased levels of LAMP-1 compared to the HeLa cells transfected with a control plasmid. Control or transfected HeLa cells were treated with U18666A (5 μg/ml) for 48–72 h, and metabolic activity ( b ) and LDH activity ( c ) were measured. Data are mean ± S.E.M. and representative of three experiments. None of transfected cell lines showed toxicity after three days of treatment with U18666A. HeLa cells lines were analyzed for cholesterol accumulation by filipin imaging ( d ). U18666A-induced cholesterol accumulation in control cells; however, LAMP-1 overexpression (HeLa-LE) suppressed U18666A-induced cholesterol accumulation. LAMP-1 knockdown (HeLa-Lkd) did not show any protection against cholesterol accumulation and showed higher accumulation of cholesterol compared to control. Microscope images are a representative of at least three random fields of three experimental replicates. Scale bar = 50 μm
    Figure Legend Snippet: LAMP-1 is essential for cyclodextrin-mediated cholesterol export from lysosomes. Verification of HeLa cells either overexpressing LAMP-1 (HeLa-LE) or stably carrying shRNA for LAMP-1 (Hela LAMP-1 knockdown or HeLa-kd) by western blot ( a ). HeLa-LE shows significantly increased levels of LAMP-1, while HeLa-Lkd shows decreased levels of LAMP-1 compared to the HeLa cells transfected with a control plasmid. Control or transfected HeLa cells were treated with U18666A (5 μg/ml) for 48–72 h, and metabolic activity ( b ) and LDH activity ( c ) were measured. Data are mean ± S.E.M. and representative of three experiments. None of transfected cell lines showed toxicity after three days of treatment with U18666A. HeLa cells lines were analyzed for cholesterol accumulation by filipin imaging ( d ). U18666A-induced cholesterol accumulation in control cells; however, LAMP-1 overexpression (HeLa-LE) suppressed U18666A-induced cholesterol accumulation. LAMP-1 knockdown (HeLa-Lkd) did not show any protection against cholesterol accumulation and showed higher accumulation of cholesterol compared to control. Microscope images are a representative of at least three random fields of three experimental replicates. Scale bar = 50 μm

    Techniques Used: Stable Transfection, shRNA, Western Blot, Transfection, Plasmid Preparation, Activity Assay, Imaging, Over Expression, Microscopy

    Effect of CD derivatives on cholesterol accumulation in NPC1 −/− cells. Primary fibroblast cells from a healthy donor or NPC patient were incubated with CD derivatives (1 mM) for 72 h and the levels of free cholesterol in cells were determined by staining with Filipin. Data shown are a representative of three independent experiments. Wild-type, primary fibroblast cells from a healthy donor; NPC1 -/- cells, primary fibroblast cells from an NPC patient. Scale bar = 50 μm
    Figure Legend Snippet: Effect of CD derivatives on cholesterol accumulation in NPC1 −/− cells. Primary fibroblast cells from a healthy donor or NPC patient were incubated with CD derivatives (1 mM) for 72 h and the levels of free cholesterol in cells were determined by staining with Filipin. Data shown are a representative of three independent experiments. Wild-type, primary fibroblast cells from a healthy donor; NPC1 -/- cells, primary fibroblast cells from an NPC patient. Scale bar = 50 μm

    Techniques Used: Incubation, Staining

    5) Product Images from "Secretion of Salmonella Pathogenicity Island 1-Encoded Type III Secretion System Effectors by Outer Membrane Vesicles in Salmonella enterica Serovar Typhimurium"

    Article Title: Secretion of Salmonella Pathogenicity Island 1-Encoded Type III Secretion System Effectors by Outer Membrane Vesicles in Salmonella enterica Serovar Typhimurium

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02810

    Translocation of SipA, SipC, and SopE2 by OMVs into the cytoplasm of the host cell. (A) OMVs-mediated translocation of SipA, SipC, and SopE2 into host cells. HeLa cells were incubated with OMVs harboring SipA-HA, SipC-HA, or SopE2-HA for 2 h. Fixed cells were treated with rabbit anti- Salmonella and mouse anti-HA antibodies. Cells were subsequently incubated with Alexa Fluor 405-conjugated anti-rabbit antibody, Alexa Fluor 488-conjugated anti-mouse antibody, and CellMask Deep Red (OMVs in blue; HA-tagged proteins in green; plasma membrane in red). Arrowheads indicate HA-tagged proteins, while arrows indicate OMVs components. The inset shows SipA-HA proximate to a vesicle in the periphery of an epithelial cell. (B) CCF4 cleavage assay for SipC-Bla translocation. HeLa cells were treated with OMVs containing SipC-Bla or not for 4 h (upper panel) or Salmonella strains (wild-type and Δ invA ) producing SipC-Bla for 3 h without gentamicin addition (lower panel). SipC-Bla translocated into the cytoplasm of host cells turned the color of CCF4 from green to blue. Cells with localized blue fluorescence are marked with arrow heads. (C) Internalization of OMVs through clathrin-mediated endocytosis. OMVs labeled with fluorescence probe R18 (R18-OMVs, red) were added to HeLa cells treated with chlorpromazine (CPZ) or filipin III (Filipin). Lipid rafts in the plasma membrane were labeled with cholera toxin B subunit (CTB) conjugated with Alexa Fluor 488 (green).
    Figure Legend Snippet: Translocation of SipA, SipC, and SopE2 by OMVs into the cytoplasm of the host cell. (A) OMVs-mediated translocation of SipA, SipC, and SopE2 into host cells. HeLa cells were incubated with OMVs harboring SipA-HA, SipC-HA, or SopE2-HA for 2 h. Fixed cells were treated with rabbit anti- Salmonella and mouse anti-HA antibodies. Cells were subsequently incubated with Alexa Fluor 405-conjugated anti-rabbit antibody, Alexa Fluor 488-conjugated anti-mouse antibody, and CellMask Deep Red (OMVs in blue; HA-tagged proteins in green; plasma membrane in red). Arrowheads indicate HA-tagged proteins, while arrows indicate OMVs components. The inset shows SipA-HA proximate to a vesicle in the periphery of an epithelial cell. (B) CCF4 cleavage assay for SipC-Bla translocation. HeLa cells were treated with OMVs containing SipC-Bla or not for 4 h (upper panel) or Salmonella strains (wild-type and Δ invA ) producing SipC-Bla for 3 h without gentamicin addition (lower panel). SipC-Bla translocated into the cytoplasm of host cells turned the color of CCF4 from green to blue. Cells with localized blue fluorescence are marked with arrow heads. (C) Internalization of OMVs through clathrin-mediated endocytosis. OMVs labeled with fluorescence probe R18 (R18-OMVs, red) were added to HeLa cells treated with chlorpromazine (CPZ) or filipin III (Filipin). Lipid rafts in the plasma membrane were labeled with cholera toxin B subunit (CTB) conjugated with Alexa Fluor 488 (green).

    Techniques Used: Translocation Assay, Incubation, Cleavage Assay, Fluorescence, Labeling, CtB Assay

    6) Product Images from "Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †"

    Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †

    Journal:

    doi: 10.1128/EC.00385-05

    The myo5 Δ and sla2 Δ mutants do not exhibit polarized lipid rafts. The wild-type and mutant cells grown under hypha-inducing conditions were stained with filipin III and visualized by epifluorescence microscopy. Bar = 5 μm.
    Figure Legend Snippet: The myo5 Δ and sla2 Δ mutants do not exhibit polarized lipid rafts. The wild-type and mutant cells grown under hypha-inducing conditions were stained with filipin III and visualized by epifluorescence microscopy. Bar = 5 μm.

    Techniques Used: Mutagenesis, Staining, Epifluorescence Microscopy

    7) Product Images from "Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes"

    Article Title: Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes

    Journal: Molecular pharmaceutics

    doi: 10.1021/mp2000428

    Effect of endocytosis inhibitors on cellular uptake of Nile Red labeled 5-8 kDa HA-liposomes (A, D) , 175-350 kDa HA-liposomes (B, E) , and fluorescein-labeled HMWHA (C, F) . Flow cytometry histogram shows the fluorescence intensities of unstained A549 cells (filled curve) and cells treated with HA-liposomes (5-8, 175-350 kDa) or fluorescein-HMWHA without inhibitor pretreatment (green curve) or with chlorpromazine (CPM, 10 μ g/mL, red curve) to inhibit clathrin-mediated endocytosis, methyl-β-cyclodextrin (MβCD, 5 mM, blue curve) to inhibit lipid raft-mediated endocytosis, amiloride (AMIL, 10 μ g/mL, brown curve) to inhibit macropinocytosis, or filipin III (5 μ g/mL, red curve) or nystatin (NYS, 25 μ g/mL, dark blue curve) to inhibit caveolae-specific endocytosis. All inhibitors were pre-incubated for 1 hour. The percentage of uptake was calculated based on the fluorescence intensity of cellular uptake without any pretreatment (100%) and is presented as mean ± SEM, n = 3-6, *** represents p
    Figure Legend Snippet: Effect of endocytosis inhibitors on cellular uptake of Nile Red labeled 5-8 kDa HA-liposomes (A, D) , 175-350 kDa HA-liposomes (B, E) , and fluorescein-labeled HMWHA (C, F) . Flow cytometry histogram shows the fluorescence intensities of unstained A549 cells (filled curve) and cells treated with HA-liposomes (5-8, 175-350 kDa) or fluorescein-HMWHA without inhibitor pretreatment (green curve) or with chlorpromazine (CPM, 10 μ g/mL, red curve) to inhibit clathrin-mediated endocytosis, methyl-β-cyclodextrin (MβCD, 5 mM, blue curve) to inhibit lipid raft-mediated endocytosis, amiloride (AMIL, 10 μ g/mL, brown curve) to inhibit macropinocytosis, or filipin III (5 μ g/mL, red curve) or nystatin (NYS, 25 μ g/mL, dark blue curve) to inhibit caveolae-specific endocytosis. All inhibitors were pre-incubated for 1 hour. The percentage of uptake was calculated based on the fluorescence intensity of cellular uptake without any pretreatment (100%) and is presented as mean ± SEM, n = 3-6, *** represents p

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

    8) Product Images from "Exploitation of the Endocytic Pathway by Orientia tsutsugamushi in Nonprofessional Phagocytes "

    Article Title: Exploitation of the Endocytic Pathway by Orientia tsutsugamushi in Nonprofessional Phagocytes

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01620-05

    Inhibitory effects of endocytosis-disrupting agents on O. tsutsugamushi invasion of nonprofessional phagocytes. ECV304 (A) and L929 (B) cells were infected at 2 × 10 5 ICU (about 15 O. tsutsugamushi bacteria were found per cell) for 2 h in the presence or absence of the inhibitors. Cells were labeled with KI-37 (monoclonal antibody against the Boryong strain's 56-kDa outer membrane protein) and goat anti-mouse IgG-FITC. The number of infecting bacteria in every 100 cells was counted by observation with a fluorescence microscope. The concentrations of inhibitors were maintained throughout the study. Each experiment was performed a minimum of three times. Inhibition assays were performed by preincubation of inhibitors for 30 min followed by O. tsutsugamushi infection for 2 h. The concentrations of inhibitors were as follows: MDC, 0.1, 0.2, 0.3, and 0.4 mM; CPZ, 1, 5, 10, and 25 μM; SUC, 0.1, 0.2, and 0.3 M; and filipin III (FIL), 0.375, 0.75, and 1.5 mM. CON, control.
    Figure Legend Snippet: Inhibitory effects of endocytosis-disrupting agents on O. tsutsugamushi invasion of nonprofessional phagocytes. ECV304 (A) and L929 (B) cells were infected at 2 × 10 5 ICU (about 15 O. tsutsugamushi bacteria were found per cell) for 2 h in the presence or absence of the inhibitors. Cells were labeled with KI-37 (monoclonal antibody against the Boryong strain's 56-kDa outer membrane protein) and goat anti-mouse IgG-FITC. The number of infecting bacteria in every 100 cells was counted by observation with a fluorescence microscope. The concentrations of inhibitors were maintained throughout the study. Each experiment was performed a minimum of three times. Inhibition assays were performed by preincubation of inhibitors for 30 min followed by O. tsutsugamushi infection for 2 h. The concentrations of inhibitors were as follows: MDC, 0.1, 0.2, 0.3, and 0.4 mM; CPZ, 1, 5, 10, and 25 μM; SUC, 0.1, 0.2, and 0.3 M; and filipin III (FIL), 0.375, 0.75, and 1.5 mM. CON, control.

    Techniques Used: Infection, Labeling, Fluorescence, Microscopy, Inhibition

    9) Product Images from "Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword"

    Article Title: Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2018.00226

    Modification of cholesterol levels in hippocampal neurons. (A,B) Quantification of filipin III fluorescence showing relative levels of cholesterol after treatment with MβCD (0.5–3 mM), and after treatment with different concentrations of soluble cholesterol (0–400 μM) in hippocampal neurons. (C,D) Cell viability assay performed after treatments to modify membrane-cholesterol and subsequent Aβ incubation (1 μM for 24 h). The bars represent the mean ± SEM. Asterisks denote: ∗ p
    Figure Legend Snippet: Modification of cholesterol levels in hippocampal neurons. (A,B) Quantification of filipin III fluorescence showing relative levels of cholesterol after treatment with MβCD (0.5–3 mM), and after treatment with different concentrations of soluble cholesterol (0–400 μM) in hippocampal neurons. (C,D) Cell viability assay performed after treatments to modify membrane-cholesterol and subsequent Aβ incubation (1 μM for 24 h). The bars represent the mean ± SEM. Asterisks denote: ∗ p

    Techniques Used: Modification, Fluorescence, Viability Assay, Incubation

    10) Product Images from "Nanotube Action between Human Mesothelial Cells Reveals Novel Aspects of Inflammatory Responses"

    Article Title: Nanotube Action between Human Mesothelial Cells Reveals Novel Aspects of Inflammatory Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029537

    Influence of MCD and simvastatin on NT formation between HPMCs. ( A ) HPMCs from Donor VI were cultured in absence or presence of indicated MCD concentrations and Filipin III staining was performed one hour after plating. Cholesterol depletion results in a decrease of fluorescence intensities (arrowheads, arrow). ( B ) Analysis of the NTs/cells ratio dependent on cholesterol depletion. HPMCs were treated with indicated MCD concentrations and NT numbers were assessed one hour after cell plating. The graph shows a significantly higher number of NTs when cells are treated with 1.25 mM MCD (arrow) as compared to the control or treatment with 0.5 mM and 2.5 mM MCD (arrowheads). ( C ) Live-cell fluorescence microscopy showing NTs (arrow) between the cells under control conditions one hour after cell plating. Scale bar: 20 µm. ( D ) Quantitative analyses of fluorescence intensities after cholesterol staining with Filipin III. Filipin staining was performed in the presence of indicated concentrations of simvastatin as described. For quantification, fluorescence intensities based on representative microscopic images (top row) were determined as described. Increasing concentrations of simvastation resulted in a linear decrease of fluorescence intensities. ( E ) Live-cell fluorescence microscopy after simvastatin treatment revealed strongly elevated NT numbers between cells (arrows) as compared to the control condition. ( F ) Quantitative analyses of the NTs/cells ratio from Donor VI after treatment with rapamycin or simvastatin. Cells were cultured in absence (control) or presence of indicated rapamycin or simvastatin concentrations and NT numbers were analyzed one hour after cell seeding. Incubation of cells with 1 nM rapamycin led to a significant decrease of NT numbers (white bar) whereas incubation with 50 µM simvastatin resulted in a strong increase (black bar). All data are means ± SEM. ** p
    Figure Legend Snippet: Influence of MCD and simvastatin on NT formation between HPMCs. ( A ) HPMCs from Donor VI were cultured in absence or presence of indicated MCD concentrations and Filipin III staining was performed one hour after plating. Cholesterol depletion results in a decrease of fluorescence intensities (arrowheads, arrow). ( B ) Analysis of the NTs/cells ratio dependent on cholesterol depletion. HPMCs were treated with indicated MCD concentrations and NT numbers were assessed one hour after cell plating. The graph shows a significantly higher number of NTs when cells are treated with 1.25 mM MCD (arrow) as compared to the control or treatment with 0.5 mM and 2.5 mM MCD (arrowheads). ( C ) Live-cell fluorescence microscopy showing NTs (arrow) between the cells under control conditions one hour after cell plating. Scale bar: 20 µm. ( D ) Quantitative analyses of fluorescence intensities after cholesterol staining with Filipin III. Filipin staining was performed in the presence of indicated concentrations of simvastatin as described. For quantification, fluorescence intensities based on representative microscopic images (top row) were determined as described. Increasing concentrations of simvastation resulted in a linear decrease of fluorescence intensities. ( E ) Live-cell fluorescence microscopy after simvastatin treatment revealed strongly elevated NT numbers between cells (arrows) as compared to the control condition. ( F ) Quantitative analyses of the NTs/cells ratio from Donor VI after treatment with rapamycin or simvastatin. Cells were cultured in absence (control) or presence of indicated rapamycin or simvastatin concentrations and NT numbers were analyzed one hour after cell seeding. Incubation of cells with 1 nM rapamycin led to a significant decrease of NT numbers (white bar) whereas incubation with 50 µM simvastatin resulted in a strong increase (black bar). All data are means ± SEM. ** p

    Techniques Used: Cell Culture, Staining, Fluorescence, Microscopy, Incubation

    Impact of MCD mediated cholesterol depletion on NT formation. ( A ) Quantitative analyses of fluorescence intensities after cholesterol staining with Filipin III in HPMCs from different donors (left panel). Corresponding fluorescence images of stained cells one hour after cell plating are shown (right panel). ( B ) HPMCs were cultured in absence or presence of indicated MCD concentrations and Filipin III staining was performed one hour after plating. For quantification, fluorescence intensities based on representative microscopic images (top row) were determined as described. Please note that cholesterol depletion results in a decrease of fluorescence intensities (arrowheads, arrow). ( C ) Analysis of the NTs/cells ratio dependent on cholesterol depletion. HPMCs were treated with indicated MCD concentrations and NT numbers were assessed one hour after cell plating. The graph shows a significantly higher number of NTs when cells are treated with 2.5 mM MCD (arrow) as compared to the control or treatment with 1.25 or 3 mM MCD (arrowheads). ( D ) Quantitative analyses of NT lengths after cholesterol depletion. HPMCs were cultured in the presence of indicated MCD concentrations and NT lengths were analyzed one hour after cell seeding. Incubation of cells with 1.25 mM MCD led to comparatively short NT lengths (arrowhead) whereas incubation with 2.5 mM MCD led to extended NT lengths (arrow). All data are means ± SEM. * p
    Figure Legend Snippet: Impact of MCD mediated cholesterol depletion on NT formation. ( A ) Quantitative analyses of fluorescence intensities after cholesterol staining with Filipin III in HPMCs from different donors (left panel). Corresponding fluorescence images of stained cells one hour after cell plating are shown (right panel). ( B ) HPMCs were cultured in absence or presence of indicated MCD concentrations and Filipin III staining was performed one hour after plating. For quantification, fluorescence intensities based on representative microscopic images (top row) were determined as described. Please note that cholesterol depletion results in a decrease of fluorescence intensities (arrowheads, arrow). ( C ) Analysis of the NTs/cells ratio dependent on cholesterol depletion. HPMCs were treated with indicated MCD concentrations and NT numbers were assessed one hour after cell plating. The graph shows a significantly higher number of NTs when cells are treated with 2.5 mM MCD (arrow) as compared to the control or treatment with 1.25 or 3 mM MCD (arrowheads). ( D ) Quantitative analyses of NT lengths after cholesterol depletion. HPMCs were cultured in the presence of indicated MCD concentrations and NT lengths were analyzed one hour after cell seeding. Incubation of cells with 1.25 mM MCD led to comparatively short NT lengths (arrowhead) whereas incubation with 2.5 mM MCD led to extended NT lengths (arrow). All data are means ± SEM. * p

    Techniques Used: Fluorescence, Staining, Cell Culture, Incubation

    11) Product Images from "Caveolin-1 and Dynamin-2 Are Essential for Removal of the Complement C5b-9 Complex via Endocytosis *"

    Article Title: Caveolin-1 and Dynamin-2 Are Essential for Removal of the Complement C5b-9 Complex via Endocytosis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.333039

    Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or filipin-III ( B ) for 30 min at 37 °C. For cholesterol
    Figure Legend Snippet: Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or filipin-III ( B ) for 30 min at 37 °C. For cholesterol

    Techniques Used: Lysis

    12) Product Images from "Early cell-autonomous accumulation of neutral lipids during infection promotes mycobacterial growth"

    Article Title: Early cell-autonomous accumulation of neutral lipids during infection promotes mycobacterial growth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0232251

    Neutral lipids accumulate cell-autonomously in M . marinum infected macrophages by 48 hours post-infection. a . Schematic diagram representing infection of 2 dpf larvae through the caudal vein, followed by cell dissociation after 48 hrs and sorting of single positive (SP, red) and double positive (DP, red-cerulean) macrophages from non-fluorescent cells. b . Epifluorescent images of representative sorted cells; non-fluorescent, red fluorescent, and red-cerulean fluorescent, respectively, from top to bottom. c . Schematic diagram representing fixation and staining of sorted cell types followed by analysis of intact cells gated on the basis of size exclusion. d . Measurement of Nile red fluorescence in sorted cell populations by analysis following excitation at 488 nm and measurement of emission via a 586/15 bandpass filter of non-fluorescent (orange), uninfected (red) and infected (cerulean) macrophages. Increases from 2.1- to 12.4- fold were observed in the geometric mean fluorescence intensity of Nile red staining between uninfected and infected macrophages. Histograms shown are the minimum and maximum fold changes observed among four independent replicates. Two additional replicates are shown in S1 Fig . e . Measurement of Filipin III fluorescence in sorted cell populations by analysis following excitation at 360 nm and measurement of emission via a 515/30 bandpass filter of non-fluorescent (orange), uninfected (red) and infected (cerulean) macrophages. No reproducible increase in fluorescence of infected macrophages compared to uninfected macrophages was observed. The histogram shown is representative of four independent replicates. Each independent experiment consisted of approximately 1000 infected larvae.
    Figure Legend Snippet: Neutral lipids accumulate cell-autonomously in M . marinum infected macrophages by 48 hours post-infection. a . Schematic diagram representing infection of 2 dpf larvae through the caudal vein, followed by cell dissociation after 48 hrs and sorting of single positive (SP, red) and double positive (DP, red-cerulean) macrophages from non-fluorescent cells. b . Epifluorescent images of representative sorted cells; non-fluorescent, red fluorescent, and red-cerulean fluorescent, respectively, from top to bottom. c . Schematic diagram representing fixation and staining of sorted cell types followed by analysis of intact cells gated on the basis of size exclusion. d . Measurement of Nile red fluorescence in sorted cell populations by analysis following excitation at 488 nm and measurement of emission via a 586/15 bandpass filter of non-fluorescent (orange), uninfected (red) and infected (cerulean) macrophages. Increases from 2.1- to 12.4- fold were observed in the geometric mean fluorescence intensity of Nile red staining between uninfected and infected macrophages. Histograms shown are the minimum and maximum fold changes observed among four independent replicates. Two additional replicates are shown in S1 Fig . e . Measurement of Filipin III fluorescence in sorted cell populations by analysis following excitation at 360 nm and measurement of emission via a 515/30 bandpass filter of non-fluorescent (orange), uninfected (red) and infected (cerulean) macrophages. No reproducible increase in fluorescence of infected macrophages compared to uninfected macrophages was observed. The histogram shown is representative of four independent replicates. Each independent experiment consisted of approximately 1000 infected larvae.

    Techniques Used: Infection, Staining, Fluorescence

    13) Product Images from "Role of surface charge in determining the biological effects of CdSe/ZnS quantum dots"

    Article Title: Role of surface charge in determining the biological effects of CdSe/ZnS quantum dots

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S94543

    The uptake pathways of different charged QDs in MDA-MB-231 cells. Notes: The effects of temperature and endocytosis inhibitors on the cellular uptake of QDs were analyzed by confocal fluorescence microscopy ( A ; 200× magnification) and flow cytometry ( B ), respectively. MDA-MB-231 Cells were either preincubated at 4°C for 1 hour, followed by incubation with different QDs at 4°C for 2 hours; or pretreated with different endocytosis inhibitors such as amiloride (50 mM), chlorpromazine (CPZ, 10 mg/mL) and filipin III (1 mg/mL) at 37°C for 1 hour, followed by incubation with different QDs at 37°C for 2 hours. Abbreviations: AMI, amiloride; CA, carboxylic acid; FIL, filipin; PDDA, polydiallydimethylammounium chloride; PEG, polyethylene glycol; QDs, quantum dots.
    Figure Legend Snippet: The uptake pathways of different charged QDs in MDA-MB-231 cells. Notes: The effects of temperature and endocytosis inhibitors on the cellular uptake of QDs were analyzed by confocal fluorescence microscopy ( A ; 200× magnification) and flow cytometry ( B ), respectively. MDA-MB-231 Cells were either preincubated at 4°C for 1 hour, followed by incubation with different QDs at 4°C for 2 hours; or pretreated with different endocytosis inhibitors such as amiloride (50 mM), chlorpromazine (CPZ, 10 mg/mL) and filipin III (1 mg/mL) at 37°C for 1 hour, followed by incubation with different QDs at 37°C for 2 hours. Abbreviations: AMI, amiloride; CA, carboxylic acid; FIL, filipin; PDDA, polydiallydimethylammounium chloride; PEG, polyethylene glycol; QDs, quantum dots.

    Techniques Used: Multiple Displacement Amplification, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Incubation

    14) Product Images from "Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives"

    Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-015-0307-4

    Two alternative routes on filipin III biosynthesis in S. filipinensis.
    Figure Legend Snippet: Two alternative routes on filipin III biosynthesis in S. filipinensis.

    Techniques Used:

    Bioassay and MICs of filipin III and its intermediates. Bioassays were performed with ethyl acetate-extracted culture broths from early stationary phase-grown cells. C. utilis was used as test organism. MICs of the purified filipin derivatives for C. utilis and S. cerevisiae were measured by broth microdilution assay (see “ Methods ”). Abbreviations are as in Fig. 4 .
    Figure Legend Snippet: Bioassay and MICs of filipin III and its intermediates. Bioassays were performed with ethyl acetate-extracted culture broths from early stationary phase-grown cells. C. utilis was used as test organism. MICs of the purified filipin derivatives for C. utilis and S. cerevisiae were measured by broth microdilution assay (see “ Methods ”). Abbreviations are as in Fig. 4 .

    Techniques Used: Purification, Microdilution Assay

    FilI inactivation increases filipin III production. a Predicted PCR fragment amplification of the parental strain and the mutant. The primers used in the assay are indicated with arrowheads . The acc(3)IV-oriT cassette is indicated in black . b PCR analysis of the wild type and the mutant. c Growth curves and time course quantification of the filipin III production in S. filipinensis ( black ) and S. filipinensis ΔfilI ( grey ).
    Figure Legend Snippet: FilI inactivation increases filipin III production. a Predicted PCR fragment amplification of the parental strain and the mutant. The primers used in the assay are indicated with arrowheads . The acc(3)IV-oriT cassette is indicated in black . b PCR analysis of the wild type and the mutant. c Growth curves and time course quantification of the filipin III production in S. filipinensis ( black ) and S. filipinensis ΔfilI ( grey ).

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis

    Filipin intermediates generated upon gene inactivation. a TLC analyses of 72 h culture broth extracts of S. filipinensis ( 4 ), S. filipinensis ΔfilC ( 2 ), S. filipinensis ΔfilD ( 3 ), and S. filipinensis ΔfilCD ( 1 ). b Relative proportions of the major filipin products accumulated by the different strains. FI, filipin I (compound Y); FII, filipin II (compound Z); FIII, filipin III; 1´-OHFI, 1´-hydroxyfillipin I (compound X).
    Figure Legend Snippet: Filipin intermediates generated upon gene inactivation. a TLC analyses of 72 h culture broth extracts of S. filipinensis ( 4 ), S. filipinensis ΔfilC ( 2 ), S. filipinensis ΔfilD ( 3 ), and S. filipinensis ΔfilCD ( 1 ). b Relative proportions of the major filipin products accumulated by the different strains. FI, filipin I (compound Y); FII, filipin II (compound Z); FIII, filipin III; 1´-OHFI, 1´-hydroxyfillipin I (compound X).

    Techniques Used: Generated, Thin Layer Chromatography

    15) Product Images from "Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response"

    Article Title: Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038733

    Squalene administration leads to accumulation of membrane cholesterol in resting CD4 T-cells. ( A ) F1 hybrid mice (n = 5/group) were injected i.p. or not (purple line) with a single dose (black line) or 4 doses of squalene (red line) within a week interval (180 µg/dose/mouse). Seven days after the last injection, negatively-sorted splenic CD4 cells from individual mice were co-stained with CD3-PE, CD4-FITC and Filipin III. Shown is the amount of cholesterol in plasma cell membrane of gated CD3 + CD4 + splenic cells as measured by MFI of Filipin III in FACS at single-cell level in one representative mouse from each group ( left panel ). Right panel , F1 hybrid mice (n = 7/group) were injected i.p. (black line) or not (red line) with a single dose of squalene (180 µg/mouse) and 7 days later negatively-sorted splenic CD4 cells from individual mice were co-stained with CD3-PE, CD4-FITC, and Filipin III. Shown is the percentage of gated CD4 + T-cells ± standard deviation (SD) and MFI values of Filipin III ± SD before and after squalene injection as collected among 700 cell events in gated population of CD3 + 4 + T-cells for one of three representative experiments. ( B ) Cholesterol accumulation in the spleen was identified by Oil Red O (ORO) staining of frozen spleen sections, counter-stained with hematoxylin from untreated or squalene treated mice (180 µg/mouse) given one or four doses, and analyzed 7 days post-injection (n = 3/group). Left panel, spleen section from untreated mouse. Middle panels, spleen section from squalene treated mice. Right panel, positive control for ORO lipid droplet staining in adipocytes. Shown is one representative ORO stained section in each group. Dark arrows indicate ORO stain. ( C ) Quantitative real-time RT-PCR of HMG-CoA reductase mRNA and Squalene epoxidase mRNA extracted from negatively-sorted CD4 splenocytes isolated from individual F1 hybrid mice (n = 5/group) that were treated (light bars) or not treated (dark bars) with a single dose of squalene (180 µg/mouse) and analyzed 7 days post-injection. Y axis indicates the mean fold increase in mRNA expression level relative to the endogenous 18S rRNA expression level (control ± standard deviation (SD). Shown are two combined separate experiments (* p value
    Figure Legend Snippet: Squalene administration leads to accumulation of membrane cholesterol in resting CD4 T-cells. ( A ) F1 hybrid mice (n = 5/group) were injected i.p. or not (purple line) with a single dose (black line) or 4 doses of squalene (red line) within a week interval (180 µg/dose/mouse). Seven days after the last injection, negatively-sorted splenic CD4 cells from individual mice were co-stained with CD3-PE, CD4-FITC and Filipin III. Shown is the amount of cholesterol in plasma cell membrane of gated CD3 + CD4 + splenic cells as measured by MFI of Filipin III in FACS at single-cell level in one representative mouse from each group ( left panel ). Right panel , F1 hybrid mice (n = 7/group) were injected i.p. (black line) or not (red line) with a single dose of squalene (180 µg/mouse) and 7 days later negatively-sorted splenic CD4 cells from individual mice were co-stained with CD3-PE, CD4-FITC, and Filipin III. Shown is the percentage of gated CD4 + T-cells ± standard deviation (SD) and MFI values of Filipin III ± SD before and after squalene injection as collected among 700 cell events in gated population of CD3 + 4 + T-cells for one of three representative experiments. ( B ) Cholesterol accumulation in the spleen was identified by Oil Red O (ORO) staining of frozen spleen sections, counter-stained with hematoxylin from untreated or squalene treated mice (180 µg/mouse) given one or four doses, and analyzed 7 days post-injection (n = 3/group). Left panel, spleen section from untreated mouse. Middle panels, spleen section from squalene treated mice. Right panel, positive control for ORO lipid droplet staining in adipocytes. Shown is one representative ORO stained section in each group. Dark arrows indicate ORO stain. ( C ) Quantitative real-time RT-PCR of HMG-CoA reductase mRNA and Squalene epoxidase mRNA extracted from negatively-sorted CD4 splenocytes isolated from individual F1 hybrid mice (n = 5/group) that were treated (light bars) or not treated (dark bars) with a single dose of squalene (180 µg/mouse) and analyzed 7 days post-injection. Y axis indicates the mean fold increase in mRNA expression level relative to the endogenous 18S rRNA expression level (control ± standard deviation (SD). Shown are two combined separate experiments (* p value

    Techniques Used: Mouse Assay, Injection, Staining, FACS, Standard Deviation, Positive Control, Quantitative RT-PCR, Isolation, Expressing

    16) Product Images from "Interaction with Caveolin-1 Modulates G Protein Coupling of Mouse ?3-Adrenoceptor *"

    Article Title: Interaction with Caveolin-1 Modulates G Protein Coupling of Mouse ?3-Adrenoceptor *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.280651

    Disruption of caveolae by filipin III alters functional responses of β 3 -AR isoforms.
    Figure Legend Snippet: Disruption of caveolae by filipin III alters functional responses of β 3 -AR isoforms.

    Techniques Used: Functional Assay

    17) Product Images from "Maintenance of CD4 T cell fitness through regulation of Foxo1"

    Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1

    Journal: Nature immunology

    doi: 10.1038/s41590-018-0157-4

    Maintained Foxo1 activity suppresses activation-induced increases in cell size and cholesterol content. a , Flow cytometric analysis of Filipin staining of isolated cultures stimulated with anti-CD3/28 ( n = 4). b , Representative confocal images of Filipin staining on day 2 of cocultures stimulated as in a . Data are representative of three independent experiments with similar results. Scale bar, 10 μM. c – e , Expression of constitutively-active STAT5b (caSTAT5b) in isolated cultures. GFP – and GFP + cells were activated for 24 h before transduction with retrovirus encoding Thy1.1 (MSCV-IRES-Thy1.1; MIT) or Thy1.1 plus caSTAT5b (MIT-caSTAT5b). Flow plots are gated on Thy1.1 + transduced cells 48 h post-transduction ( c , n = 7; d , n = 9; e , n = 7). f – h , Myc overexpression in isolated cultures. GFP – and GFP + cells were activated for 24 h before transduction with retrovirus encoding RFP (MSCV-IRES-RFP; MIR) or RFP plus c-Myc (MIR-Myc). Flow plots are gated on RFP + -transduced cells 48 h post-transduction. Analysis of Filipin staining was subsequent to sorting RFP + populations ( f , n = 3; g , n = 6; h , n = 4). For all graphs, quantification involved a two-tailed Student’s t test with no adjustments made for multiple comparisons; center value, mean; error bars, s.d. * P
    Figure Legend Snippet: Maintained Foxo1 activity suppresses activation-induced increases in cell size and cholesterol content. a , Flow cytometric analysis of Filipin staining of isolated cultures stimulated with anti-CD3/28 ( n = 4). b , Representative confocal images of Filipin staining on day 2 of cocultures stimulated as in a . Data are representative of three independent experiments with similar results. Scale bar, 10 μM. c – e , Expression of constitutively-active STAT5b (caSTAT5b) in isolated cultures. GFP – and GFP + cells were activated for 24 h before transduction with retrovirus encoding Thy1.1 (MSCV-IRES-Thy1.1; MIT) or Thy1.1 plus caSTAT5b (MIT-caSTAT5b). Flow plots are gated on Thy1.1 + transduced cells 48 h post-transduction ( c , n = 7; d , n = 9; e , n = 7). f – h , Myc overexpression in isolated cultures. GFP – and GFP + cells were activated for 24 h before transduction with retrovirus encoding RFP (MSCV-IRES-RFP; MIR) or RFP plus c-Myc (MIR-Myc). Flow plots are gated on RFP + -transduced cells 48 h post-transduction. Analysis of Filipin staining was subsequent to sorting RFP + populations ( f , n = 3; g , n = 6; h , n = 4). For all graphs, quantification involved a two-tailed Student’s t test with no adjustments made for multiple comparisons; center value, mean; error bars, s.d. * P

    Techniques Used: Activity Assay, Activation Assay, Flow Cytometry, Staining, Isolation, Expressing, Transduction, Over Expression, Two Tailed Test

    Cholesterol-dependent immunological synapse formation is disrupted by maintained Foxo1 activity. a , Representative total internal reflection fluorescence microscope images of mouse CD4 T cells expressing wild-type or constitutively active Foxo1 with or without water-soluble cholesterol supplementation, interacting with SLBs containing fluorescent ICAM-1 and CD80, and anti-TCR 2C11. SLB-bound T cell surface was visualized by interference reflection microscopy (IRM). Data are representative of three independent experiments with similar results. Scale bar, 5 μM. b , Quantification of immunological synapse formation by T cells, as a percentage of all SLB-bound T cells per experiment. Successful formation was defined by the appearance of a highly dense 2C11 area excluding ICAM-1 at the site of SLB interaction (wild type, n = 6 cultures; AAA, n = 6; AAA + cholesterol (chol.), n = 3). c – e , Quantification of TCR and LFA-1 recruitment, as determined by integrated density (int. dens.) of 2C11 and ICAM-1, respectively. IRM was used to determine T cell spreading, and area was used as a mask for quantification of integrated fluorescence density. K denotes thousands. f , Cellular levels of cholesterol, determined by Filipin staining. Graphs indicate percentage or mean ± s.e.m. One-way analysis of variance was performed on all datasets and indicated P
    Figure Legend Snippet: Cholesterol-dependent immunological synapse formation is disrupted by maintained Foxo1 activity. a , Representative total internal reflection fluorescence microscope images of mouse CD4 T cells expressing wild-type or constitutively active Foxo1 with or without water-soluble cholesterol supplementation, interacting with SLBs containing fluorescent ICAM-1 and CD80, and anti-TCR 2C11. SLB-bound T cell surface was visualized by interference reflection microscopy (IRM). Data are representative of three independent experiments with similar results. Scale bar, 5 μM. b , Quantification of immunological synapse formation by T cells, as a percentage of all SLB-bound T cells per experiment. Successful formation was defined by the appearance of a highly dense 2C11 area excluding ICAM-1 at the site of SLB interaction (wild type, n = 6 cultures; AAA, n = 6; AAA + cholesterol (chol.), n = 3). c – e , Quantification of TCR and LFA-1 recruitment, as determined by integrated density (int. dens.) of 2C11 and ICAM-1, respectively. IRM was used to determine T cell spreading, and area was used as a mask for quantification of integrated fluorescence density. K denotes thousands. f , Cellular levels of cholesterol, determined by Filipin staining. Graphs indicate percentage or mean ± s.e.m. One-way analysis of variance was performed on all datasets and indicated P

    Techniques Used: Activity Assay, Fluorescence, Microscopy, Expressing, Staining

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

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

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M043299

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

    Techniques Used: Staining

    19) Product Images from "Evolution of availability of curcumin inside poly-lactic-co-glycolic acid nanoparticles: impact on antioxidant and antinitrosant properties"

    Article Title: Evolution of availability of curcumin inside poly-lactic-co-glycolic acid nanoparticles: impact on antioxidant and antinitrosant properties

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S84760

    Mechanism of potentiation of antioxidant activity by PLGA-NP. Notes: A549 cells were treated with endocytosis pathway (Fil, Nys, PAO, Chl) or efflux pump (Ela) inhibitors then loaded with free curcumin ( A ) or PLGA/DiD-NP ( B ). The fluorescence was analyzed by cytometry and results are expressed as mean fluorescence intensity ± SEM. Abbreviations: PLGA, poly-lactic-co-glycolic acid; NP, nanoparticles; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; SEM, standard error of the mean; Fil, filipin III; Nys, nystatin; PAO, phenylarsine oxide; Chl, chlorpromazine; Ela, elacridar.
    Figure Legend Snippet: Mechanism of potentiation of antioxidant activity by PLGA-NP. Notes: A549 cells were treated with endocytosis pathway (Fil, Nys, PAO, Chl) or efflux pump (Ela) inhibitors then loaded with free curcumin ( A ) or PLGA/DiD-NP ( B ). The fluorescence was analyzed by cytometry and results are expressed as mean fluorescence intensity ± SEM. Abbreviations: PLGA, poly-lactic-co-glycolic acid; NP, nanoparticles; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; SEM, standard error of the mean; Fil, filipin III; Nys, nystatin; PAO, phenylarsine oxide; Chl, chlorpromazine; Ela, elacridar.

    Techniques Used: Antioxidant Activity Assay, Fluorescence, Cytometry

    20) Product Images from "Cholesterol-dependent clustering of IL-2R? and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts"

    Article Title: Cholesterol-dependent clustering of IL-2R? and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Cluster sizes of IL-2Rα, HLA class I and II, CD48, and TrfR and their modulation by membrane cholesterol content. Cluster sizes on Kit 225 K6 cells determined from the angle-averaged autocorrelation function are presented for IL-2Rα (filled columns), HLA class I (crosshatched columns), and class II (striped columns), CD48 (gray columns), and TrfR (open columns). The effect of modulating the cholesterol content of the membrane is also displayed: with the exception of TrfR, all receptor clusters exhibit a significant increase of cluster size on both cholesterol depletion by cyclodextrin or in situ complexation of cholesterol by filipin. ( n > 9, from three independent experiments).
    Figure Legend Snippet: Cluster sizes of IL-2Rα, HLA class I and II, CD48, and TrfR and their modulation by membrane cholesterol content. Cluster sizes on Kit 225 K6 cells determined from the angle-averaged autocorrelation function are presented for IL-2Rα (filled columns), HLA class I (crosshatched columns), and class II (striped columns), CD48 (gray columns), and TrfR (open columns). The effect of modulating the cholesterol content of the membrane is also displayed: with the exception of TrfR, all receptor clusters exhibit a significant increase of cluster size on both cholesterol depletion by cyclodextrin or in situ complexation of cholesterol by filipin. ( n > 9, from three independent experiments).

    Techniques Used: In Situ

    21) Product Images from "?v?6- and ?v?8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion"

    Article Title: ?v?6- and ?v?8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003806

    Effect of expression of αvβ6- or αvβ8-integrin on the pathway of HSV entry, defined through sensitivity to specific inhibitors. J cells were transfected with plasmids encoding nectin1 (black diamond), nectin1 plus αvβ3-integrin (pink square), nectin1 plus αvβ6-integrin (red triangle), or nectin1 plus αvβ8-integrin (green circle). 48 h after transfection cells were pretreated with increasing amounts of BFLA (A), wortmannin (B), filipin III (C) or dynasore (D) and infected with R8102 (3 pfu/cell) for 90 min at 37°C in the same medium. Inoculum was removed and cells were overlaid with Dulbecco's minimal essential medium (DMEM) for 6–8 h. Specific details were as follows. For filipin III and dynasore, cells were preincubated with the compounds at 37°C for 30 min or 60 min, respectively, and infected for 30 min (30 pfu/cell) in the same medium. Infected cells were overlaid without inhibitor. BFLA or wortmannin were present also after virus absorption. Infection was quantified as detailed in the legend to Fig. 3 , and expressed as percentage of untreated cells. Bars show SD.
    Figure Legend Snippet: Effect of expression of αvβ6- or αvβ8-integrin on the pathway of HSV entry, defined through sensitivity to specific inhibitors. J cells were transfected with plasmids encoding nectin1 (black diamond), nectin1 plus αvβ3-integrin (pink square), nectin1 plus αvβ6-integrin (red triangle), or nectin1 plus αvβ8-integrin (green circle). 48 h after transfection cells were pretreated with increasing amounts of BFLA (A), wortmannin (B), filipin III (C) or dynasore (D) and infected with R8102 (3 pfu/cell) for 90 min at 37°C in the same medium. Inoculum was removed and cells were overlaid with Dulbecco's minimal essential medium (DMEM) for 6–8 h. Specific details were as follows. For filipin III and dynasore, cells were preincubated with the compounds at 37°C for 30 min or 60 min, respectively, and infected for 30 min (30 pfu/cell) in the same medium. Infected cells were overlaid without inhibitor. BFLA or wortmannin were present also after virus absorption. Infection was quantified as detailed in the legend to Fig. 3 , and expressed as percentage of untreated cells. Bars show SD.

    Techniques Used: Expressing, Transfection, Infection

    22) Product Images from "2-Hydroxypropyl-gamma-cyclodextrin overcomes NPC1 deficiency by enhancing lysosome-ER association and autophagy"

    Article Title: 2-Hydroxypropyl-gamma-cyclodextrin overcomes NPC1 deficiency by enhancing lysosome-ER association and autophagy

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-65627-4

    HPγCD alleviates cholesterol accumulation in NPC1 fibroblasts. Skin fibroblasts from a healthy donor (Healthy) or NPC1 patient (NPC1) were treated for 72 h with HPβCD (1 mM) or HPγCD (1 mM) and then examined for cholesterol and neutral lipid content. The levels of cholesterol in cell lysates were measured by cholesterol assay ( a , b ) in a reaction mixture with (for total cholesterol content) or without (for free cholesterol content) cholesterol esterase enzyme. The intracellular accumulation of free cholesterol and neutral lipid were evaluated by staining with Filipin and Nile Red, respectively ( c ). Data are mean ± S.E.M. of triplicates and a representative of three independent experiments. Symbols indicate the relative level of significance compared with the control (***P
    Figure Legend Snippet: HPγCD alleviates cholesterol accumulation in NPC1 fibroblasts. Skin fibroblasts from a healthy donor (Healthy) or NPC1 patient (NPC1) were treated for 72 h with HPβCD (1 mM) or HPγCD (1 mM) and then examined for cholesterol and neutral lipid content. The levels of cholesterol in cell lysates were measured by cholesterol assay ( a , b ) in a reaction mixture with (for total cholesterol content) or without (for free cholesterol content) cholesterol esterase enzyme. The intracellular accumulation of free cholesterol and neutral lipid were evaluated by staining with Filipin and Nile Red, respectively ( c ). Data are mean ± S.E.M. of triplicates and a representative of three independent experiments. Symbols indicate the relative level of significance compared with the control (***P

    Techniques Used: Cholesterol Assay, Staining

    HPγCD promotes TFEB activation in NPC1 fibroblasts. TFEB expression, activation and the induction of TFEB target (the CLEAR network) were evaluated in NPC1 fibroblasts following the treatment without or with HPγCD (1 mM, 48 h). Cells were lysed and immunoblotted for TFEB ( a ). The blots are from different parts of the same gel and delineated with dividing lines. The HPγCD-treated cells showed significantly higher TFEB protein levels as calculated by densitometry analysis. TFEB activation was evaluated by nuclear localization of TFEB as analyzed by confocal microscopy ( b ). Microscopic images showed nuclear localization of TFEB as a purple color (merge) resulted from co-localization of TFEB (red) and DAPI (blue). The co-localization was measured by Pearson’s coefficient. Real-time PCR was used to analyze the relative mRNA expression levels of TFEB target genes in NPC1 mutant cells following the treatment without or with HPγCD (1 mM, 48 h). The expression levels of the members of CLEAR gene network (CTSB, CLCN7 and PSAP) were calculated by considering GAPDH as a reference gene and data was presented as fold changes in expression as compared to untreated cells ( c ). The effect of genistein (GNT; 25 μM, 48 h) on intracellular accumulation of free cholesterol in NPC1 mutant cells was evaluated by staining with Filipin ( d ). Data are mean ± S.E.M. of triplicates and a representative of three independent experiments. Symbols indicate the relative level of significance compared with the control (**P
    Figure Legend Snippet: HPγCD promotes TFEB activation in NPC1 fibroblasts. TFEB expression, activation and the induction of TFEB target (the CLEAR network) were evaluated in NPC1 fibroblasts following the treatment without or with HPγCD (1 mM, 48 h). Cells were lysed and immunoblotted for TFEB ( a ). The blots are from different parts of the same gel and delineated with dividing lines. The HPγCD-treated cells showed significantly higher TFEB protein levels as calculated by densitometry analysis. TFEB activation was evaluated by nuclear localization of TFEB as analyzed by confocal microscopy ( b ). Microscopic images showed nuclear localization of TFEB as a purple color (merge) resulted from co-localization of TFEB (red) and DAPI (blue). The co-localization was measured by Pearson’s coefficient. Real-time PCR was used to analyze the relative mRNA expression levels of TFEB target genes in NPC1 mutant cells following the treatment without or with HPγCD (1 mM, 48 h). The expression levels of the members of CLEAR gene network (CTSB, CLCN7 and PSAP) were calculated by considering GAPDH as a reference gene and data was presented as fold changes in expression as compared to untreated cells ( c ). The effect of genistein (GNT; 25 μM, 48 h) on intracellular accumulation of free cholesterol in NPC1 mutant cells was evaluated by staining with Filipin ( d ). Data are mean ± S.E.M. of triplicates and a representative of three independent experiments. Symbols indicate the relative level of significance compared with the control (**P

    Techniques Used: Activation Assay, Expressing, Confocal Microscopy, Real-time Polymerase Chain Reaction, Mutagenesis, Staining

    23) Product Images from "Non-Canonical Activation of the Epidermal Growth Factor Receptor by Carbon Nanoparticles"

    Article Title: Non-Canonical Activation of the Epidermal Growth Factor Receptor by Carbon Nanoparticles

    Journal: Nanomaterials

    doi: 10.3390/nano8040267

    Caveolin-1 protein complexes in dithiobis-succimidylpropionate (DSP)-cross-linked protein extracts. After exposure RLE-6TN cells were treated with DSP (1 mM, 1 h at 4 °C) to stabilize higher order caveolin-1 structures to be detectable by Western blotting. Means and standard errors as well as representative Western-blots are depicted. ( A ) Cells were exposed (5 min) to CNP (10 μg/cm 2 ), EGF (100 ng/mL), or C6 ceramide (5 μM). Cell were pre-treated with (18 h) N -acetylcysteine (NAC, 1 mM); ( B ), or 1 h with α-tocopherol (Toco, 75 µM) ( C ), or filipin III (Fil, 1 µg/mL) ( D ). *, significantly different to PBS control ( p
    Figure Legend Snippet: Caveolin-1 protein complexes in dithiobis-succimidylpropionate (DSP)-cross-linked protein extracts. After exposure RLE-6TN cells were treated with DSP (1 mM, 1 h at 4 °C) to stabilize higher order caveolin-1 structures to be detectable by Western blotting. Means and standard errors as well as representative Western-blots are depicted. ( A ) Cells were exposed (5 min) to CNP (10 μg/cm 2 ), EGF (100 ng/mL), or C6 ceramide (5 μM). Cell were pre-treated with (18 h) N -acetylcysteine (NAC, 1 mM); ( B ), or 1 h with α-tocopherol (Toco, 75 µM) ( C ), or filipin III (Fil, 1 µg/mL) ( D ). *, significantly different to PBS control ( p

    Techniques Used: Western Blot

    Caveolin-1 and EGFR co-localization as a feature of non-canonical EGFR activation. Epithelial cells (RLE-6TN) were exposed (5 min) to carbon nanoparticles (CNP), non-nano carbon particles (CP), each 10 µg/cm 2 , (50 µM) H 2 O 2 , (5 µM) C6 ceramide, or EGF (100 ng/mL), respectively. ( A ) Subcellular localization of EGFR (red Alexa flour 594) and caveolin-1 (green Alexa flour 488). Co-localization is visualized by the yellow color in merged images; ( B ) Subcellular localization of EGFR and caveolin-1 in cells pre-treated with inhibitors of carbon nanoparticle-specific signaling prior to particle or EGF exposure: SFK inhibitor PP2 (10 µM), 1 mM ectoine ( E ), 75 µM α-tocopherol (Toco), and 1 µg/mL filipin III (Fil). Co-localization is visualized by the yellow color in merged images; ( C ) Subcellular localization of caveolin-1 (red Alexa fluor 594) after exposure to carbon nanoparticles (CNP), carbon particles (CP), or hydrogen peroxide (H 2 O 2 ); ( D ) Quantification and representative Western blots of caveolin-1 in lipid raft fraction of RLE-6TN cells exposed to CNP (10 µg/cm 2 ). Raft and non-raft fraction were isolated from density gradients after ultracentrifugation. Pre-treatment of cells with α-tocopherol (Toco) was applied as an antioxidant strategy. GAPDH was used as a control protein not associated with lipid rafts. The bars in the graph represent the additive immune signals of raft and non-raft fractions, which was indicated in the representative original Western blots; ( E ) Quantification and representative Western-blot of EGFR phosphorylation at Tyr 845 . Nuclei were stained with DAPI (blue). Scale bars represent 20 µm; *, which was significantly different to PBS control ( p
    Figure Legend Snippet: Caveolin-1 and EGFR co-localization as a feature of non-canonical EGFR activation. Epithelial cells (RLE-6TN) were exposed (5 min) to carbon nanoparticles (CNP), non-nano carbon particles (CP), each 10 µg/cm 2 , (50 µM) H 2 O 2 , (5 µM) C6 ceramide, or EGF (100 ng/mL), respectively. ( A ) Subcellular localization of EGFR (red Alexa flour 594) and caveolin-1 (green Alexa flour 488). Co-localization is visualized by the yellow color in merged images; ( B ) Subcellular localization of EGFR and caveolin-1 in cells pre-treated with inhibitors of carbon nanoparticle-specific signaling prior to particle or EGF exposure: SFK inhibitor PP2 (10 µM), 1 mM ectoine ( E ), 75 µM α-tocopherol (Toco), and 1 µg/mL filipin III (Fil). Co-localization is visualized by the yellow color in merged images; ( C ) Subcellular localization of caveolin-1 (red Alexa fluor 594) after exposure to carbon nanoparticles (CNP), carbon particles (CP), or hydrogen peroxide (H 2 O 2 ); ( D ) Quantification and representative Western blots of caveolin-1 in lipid raft fraction of RLE-6TN cells exposed to CNP (10 µg/cm 2 ). Raft and non-raft fraction were isolated from density gradients after ultracentrifugation. Pre-treatment of cells with α-tocopherol (Toco) was applied as an antioxidant strategy. GAPDH was used as a control protein not associated with lipid rafts. The bars in the graph represent the additive immune signals of raft and non-raft fractions, which was indicated in the representative original Western blots; ( E ) Quantification and representative Western-blot of EGFR phosphorylation at Tyr 845 . Nuclei were stained with DAPI (blue). Scale bars represent 20 µm; *, which was significantly different to PBS control ( p

    Techniques Used: Activation Assay, Western Blot, Isolation, Staining

    24) Product Images from "Fluorescent Analysis of the Cell-Selective Alzheimer's Disease Aβ Peptide Surface Membrane Binding: Influence of Membrane Components"

    Article Title: Fluorescent Analysis of the Cell-Selective Alzheimer's Disease Aβ Peptide Surface Membrane Binding: Influence of Membrane Components

    Journal: International Journal of Alzheimer's Disease

    doi: 10.4061/2011/917629

    Correlation between the levels of cholesterol in the membrane and the binding of A β -FAM to the membrane surface. PC12 cells were exposed for 80 minutes to A β 42-FAM, washed to remove unbound A β -FAM, and then incubated for 40 minutes in PBS containing Filipin III. (a) shows the flow cytometric analysis of the cholesterol content of the three subpopulations of cells distinguished on the bases of the level of A β -FAM binding. The percentage of cells and the Geo mean of fluorescence (GeoMF) values measured within the M gate are displayed in the bottom table. The highest level of filipin III fluorescence is observed in the cells from the subpopulation C, defined as extra high A β -FAM affinity. The bars plot in (b) summarizes the percentage of cells and the GMF values within gate M corresponding to each subpopulation of data pooled from four experiments.
    Figure Legend Snippet: Correlation between the levels of cholesterol in the membrane and the binding of A β -FAM to the membrane surface. PC12 cells were exposed for 80 minutes to A β 42-FAM, washed to remove unbound A β -FAM, and then incubated for 40 minutes in PBS containing Filipin III. (a) shows the flow cytometric analysis of the cholesterol content of the three subpopulations of cells distinguished on the bases of the level of A β -FAM binding. The percentage of cells and the Geo mean of fluorescence (GeoMF) values measured within the M gate are displayed in the bottom table. The highest level of filipin III fluorescence is observed in the cells from the subpopulation C, defined as extra high A β -FAM affinity. The bars plot in (b) summarizes the percentage of cells and the GMF values within gate M corresponding to each subpopulation of data pooled from four experiments.

    Techniques Used: Binding Assay, Incubation, Flow Cytometry, Fluorescence

    25) Product Images from "High lipid order of Arabidopsis cell-plate membranes mediated by sterol and DYNAMIN-RELATED PROTEIN1A function"

    Article Title: High lipid order of Arabidopsis cell-plate membranes mediated by sterol and DYNAMIN-RELATED PROTEIN1A function

    Journal: The Plant Journal

    doi: 10.1111/tpj.12674

    DRP1A is enriched in DRMs and co-localizes with sterols at the cell plate.(a–d) Western blot analysis of DRM fractions from 3-week-old Arabidopsis callus cultures of wild-type L er and the cpi1 - 1 mutant (in the L er background). Equal amounts of membrane protein were loaded from the control fraction mock-extracted at a Triton X-100 detergent/protein (w/w) ratio of 0 (non-DRM) and the DRM fraction extracted at a Triton X-100 detergent/protein (w/w) ratio of 8 (DRM). Similar results were obtained in three independent experiments.(a) Western blot from DRM extractions probed with anti-DRP1A (isoform-specific), anti-CLC (generic), anti-ARF1 (generic) and anti-SMT1 (isoform-specific) antibodies.(b) Replicate Coomassie Blue gel as a loading control for the blot in (a).(c) Western blot from DRM extraction probed with anti-CHC (generic) and anti-SMT1 (isoform-specific) antibodies.(d) Replicate Coomassie Blue gel as a loading control for the blot in (c).The results in (a) and (c) indicate enrichment of DRP1A, CLC2, CHC and ARF1 in DRMs compared to depleted SMT1.(e–m) Co-localization analyses at the cell plate in late cytokinetic cells: (e,h,k) filipin-sterol fluorescence (fil, red); (f) DRP1A–GFP, (i) DRP2B–GFP and (l) CLC2–GFP fluorescence (green). (g,j,m) Merged images of (e) and (f), (h) and (i), and (k) and (l), respectively. Scale bars = 5 μm.
    Figure Legend Snippet: DRP1A is enriched in DRMs and co-localizes with sterols at the cell plate.(a–d) Western blot analysis of DRM fractions from 3-week-old Arabidopsis callus cultures of wild-type L er and the cpi1 - 1 mutant (in the L er background). Equal amounts of membrane protein were loaded from the control fraction mock-extracted at a Triton X-100 detergent/protein (w/w) ratio of 0 (non-DRM) and the DRM fraction extracted at a Triton X-100 detergent/protein (w/w) ratio of 8 (DRM). Similar results were obtained in three independent experiments.(a) Western blot from DRM extractions probed with anti-DRP1A (isoform-specific), anti-CLC (generic), anti-ARF1 (generic) and anti-SMT1 (isoform-specific) antibodies.(b) Replicate Coomassie Blue gel as a loading control for the blot in (a).(c) Western blot from DRM extraction probed with anti-CHC (generic) and anti-SMT1 (isoform-specific) antibodies.(d) Replicate Coomassie Blue gel as a loading control for the blot in (c).The results in (a) and (c) indicate enrichment of DRP1A, CLC2, CHC and ARF1 in DRMs compared to depleted SMT1.(e–m) Co-localization analyses at the cell plate in late cytokinetic cells: (e,h,k) filipin-sterol fluorescence (fil, red); (f) DRP1A–GFP, (i) DRP2B–GFP and (l) CLC2–GFP fluorescence (green). (g,j,m) Merged images of (e) and (f), (h) and (i), and (k) and (l), respectively. Scale bars = 5 μm.

    Techniques Used: Western Blot, Mutagenesis, Fluorescence

    26) Product Images from "Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives"

    Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-015-0307-4

    Two alternative routes on filipin III biosynthesis in S. filipinensis.
    Figure Legend Snippet: Two alternative routes on filipin III biosynthesis in S. filipinensis.

    Techniques Used:

    Bioassay and MICs of filipin III and its intermediates. Bioassays were performed with ethyl acetate-extracted culture broths from early stationary phase-grown cells. C. utilis was used as test organism. MICs of the purified filipin derivatives for C. utilis and S. cerevisiae were measured by broth microdilution assay (see “ Methods ”). Abbreviations are as in Fig. 4 .
    Figure Legend Snippet: Bioassay and MICs of filipin III and its intermediates. Bioassays were performed with ethyl acetate-extracted culture broths from early stationary phase-grown cells. C. utilis was used as test organism. MICs of the purified filipin derivatives for C. utilis and S. cerevisiae were measured by broth microdilution assay (see “ Methods ”). Abbreviations are as in Fig. 4 .

    Techniques Used: Purification, Microdilution Assay

    FilI inactivation increases filipin III production. a Predicted PCR fragment amplification of the parental strain and the mutant. The primers used in the assay are indicated with arrowheads . The acc(3)IV-oriT cassette is indicated in black . b PCR analysis of the wild type and the mutant. c Growth curves and time course quantification of the filipin III production in S. filipinensis ( black ) and S. filipinensis ΔfilI ( grey ).
    Figure Legend Snippet: FilI inactivation increases filipin III production. a Predicted PCR fragment amplification of the parental strain and the mutant. The primers used in the assay are indicated with arrowheads . The acc(3)IV-oriT cassette is indicated in black . b PCR analysis of the wild type and the mutant. c Growth curves and time course quantification of the filipin III production in S. filipinensis ( black ) and S. filipinensis ΔfilI ( grey ).

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis

    Filipin intermediates generated upon gene inactivation. a TLC analyses of 72 h culture broth extracts of S. filipinensis ( 4 ), S. filipinensis ΔfilC ( 2 ), S. filipinensis ΔfilD ( 3 ), and S. filipinensis ΔfilCD ( 1 ). b Relative proportions of the major filipin products accumulated by the different strains. FI, filipin I (compound Y); FII, filipin II (compound Z); FIII, filipin III; 1´-OHFI, 1´-hydroxyfillipin I (compound X).
    Figure Legend Snippet: Filipin intermediates generated upon gene inactivation. a TLC analyses of 72 h culture broth extracts of S. filipinensis ( 4 ), S. filipinensis ΔfilC ( 2 ), S. filipinensis ΔfilD ( 3 ), and S. filipinensis ΔfilCD ( 1 ). b Relative proportions of the major filipin products accumulated by the different strains. FI, filipin I (compound Y); FII, filipin II (compound Z); FIII, filipin III; 1´-OHFI, 1´-hydroxyfillipin I (compound X).

    Techniques Used: Generated, Thin Layer Chromatography

    27) Product Images from "Involvement of caveolin-1 in the Jak–Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish (Siniperca chuatsi)"

    Article Title: Involvement of caveolin-1 in the Jak–Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish (Siniperca chuatsi)

    Journal: Molecular Immunology

    doi: 10.1016/j.molimm.2011.01.001

    Subcellular distribution of mCav-1 protein in MFF-1 cells. (A) Immunofluorescence analysis of mCav-1 protein in MFF-1 cells. The red fluorescence was immunostained by anti-mCav-1 antiserum, and the blue fluorescence was immunostained by Hoechst 33342. Magnification: ×400. (B) Western blot analysis of mCav-1 protein. Cholesterol-rich fractions were isolated by 1% Triton X-100 treatment and ultracentrifugation in a sucrose gradient. After centrifugation, the total proteins in each fraction were separated by gel electrophoresis and immunoblotted with anti-mCav-1 antiserum. Lanes 1–12 are sucrose gradient fractions (top to bottom); lanes 4–9 are the DRM (detergent-resistant membrane) fractions (caveolae fragments); lanes 10–12 are the DSM (detergent-soluble membrane) fractions (cytoplasm fragments). (C) MβCD, filipin III, and nystatin altered the distribution of mCav-1 protein. Cells either were not treated or were incubated in the presence of MβCD, filipin III, or nystatin for 60 min before isolation of the cholesterol-rich fractions. Each fraction was immunoblotted with anti-mCav-1 antiserum. Lanes 1–12 are the sucrose gradients fractions (top to bottom); lanes 4–9 are the DRM fractions; lanes 10–12 are the DSM fractions; the last lane is lysate from MFF-1 cells before extraction of cholesterol-rich fractions. (D) Electron microscopy. Caveolae were identified and observed close to the plasma membrane in MFF-1 cells (arrows). Scale bars: 800 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
    Figure Legend Snippet: Subcellular distribution of mCav-1 protein in MFF-1 cells. (A) Immunofluorescence analysis of mCav-1 protein in MFF-1 cells. The red fluorescence was immunostained by anti-mCav-1 antiserum, and the blue fluorescence was immunostained by Hoechst 33342. Magnification: ×400. (B) Western blot analysis of mCav-1 protein. Cholesterol-rich fractions were isolated by 1% Triton X-100 treatment and ultracentrifugation in a sucrose gradient. After centrifugation, the total proteins in each fraction were separated by gel electrophoresis and immunoblotted with anti-mCav-1 antiserum. Lanes 1–12 are sucrose gradient fractions (top to bottom); lanes 4–9 are the DRM (detergent-resistant membrane) fractions (caveolae fragments); lanes 10–12 are the DSM (detergent-soluble membrane) fractions (cytoplasm fragments). (C) MβCD, filipin III, and nystatin altered the distribution of mCav-1 protein. Cells either were not treated or were incubated in the presence of MβCD, filipin III, or nystatin for 60 min before isolation of the cholesterol-rich fractions. Each fraction was immunoblotted with anti-mCav-1 antiserum. Lanes 1–12 are the sucrose gradients fractions (top to bottom); lanes 4–9 are the DRM fractions; lanes 10–12 are the DSM fractions; the last lane is lysate from MFF-1 cells before extraction of cholesterol-rich fractions. (D) Electron microscopy. Caveolae were identified and observed close to the plasma membrane in MFF-1 cells (arrows). Scale bars: 800 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

    Techniques Used: Immunofluorescence, Fluorescence, Western Blot, Isolation, Centrifugation, Nucleic Acid Electrophoresis, Incubation, Electron Microscopy

    28) Product Images from "Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis"

    Article Title: Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

    Journal: Virology

    doi: 10.1016/j.virol.2008.04.041

    Influence of cholesterol depletion on EAV infection of BHK-21 cells. (A) After pretreatment of BHK cells with MßCD or (B) incubation with filipin III infection of cells by EAV (filled circles) or by VSV (open circles) was assessed by the plaque assay. For treatment of the cells with substances see Materials and methods . Data present mean ± standard error of estimate of three independent experiments.
    Figure Legend Snippet: Influence of cholesterol depletion on EAV infection of BHK-21 cells. (A) After pretreatment of BHK cells with MßCD or (B) incubation with filipin III infection of cells by EAV (filled circles) or by VSV (open circles) was assessed by the plaque assay. For treatment of the cells with substances see Materials and methods . Data present mean ± standard error of estimate of three independent experiments.

    Techniques Used: Infection, Incubation, Plaque Assay

    29) Product Images from "Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation"

    Article Title: Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011014

    Effects of Filipin III and monodansylcadaverine on LRP6 turnover. HT1080 cells stably transduced with HA-tagged LRP6 were incubated with 0.5 µg/ml of recombinant Dkk1 protein (A, C) or 25% of Wnt3A CM (B, D), and treated with Filipin III (3 µg/ml) (A, B) or monodansylcadaverine (MDC) (100 µM) for 24 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. Samples were also probed with the anti-actin antibody to verify equal loading.
    Figure Legend Snippet: Effects of Filipin III and monodansylcadaverine on LRP6 turnover. HT1080 cells stably transduced with HA-tagged LRP6 were incubated with 0.5 µg/ml of recombinant Dkk1 protein (A, C) or 25% of Wnt3A CM (B, D), and treated with Filipin III (3 µg/ml) (A, B) or monodansylcadaverine (MDC) (100 µM) for 24 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. Samples were also probed with the anti-actin antibody to verify equal loading.

    Techniques Used: Stable Transfection, Transduction, Incubation, Recombinant, Western Blot

    30) 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

    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

    ). Representative images for each time point after infection are depicted. The areas highlighted with white rectangles in the middle panels are shown in the cropped images below. Therein two areas (a and b) are highlighted, which are shown as enlargements in the right panel. The adjacent histograms indicate the signal intensity profiles of NS5A and cholesterol along the respective arrow. Bars, 10 μm. (B) Quantification of time-dependent changes of cholesterol distribution patterns, classified into the following groups: (i) cholesterol mainly present at the plasma membrane, (ii) cholesterol mainly distributed in a vesicular pattern, and (iii) cholesterol accumulating in a web-like diffuse perinuclear structure. Results for at least two independent experiments are shown ( n , total number of cells per condition). (C) Quantification of the degrees of colocalization (Manders overlap coefficients) of NS5A and filipin at 24, 48, and 96 h postinfection. Results for two independent experiments are shown; the total number of cells per condition was ≥36. (D) Quantification of the filipin signals at 24, 48, and 72 h postinfection. The integrated fluorescence densities of the filipin signals in NS5A-positive or -negative (no virus) cells were quantified and are given in arbitrary units (AU). Results of two representative experiments are shown. The number of cells per condition was ≥11. ***, P
    Figure Legend Snippet: ). Representative images for each time point after infection are depicted. The areas highlighted with white rectangles in the middle panels are shown in the cropped images below. Therein two areas (a and b) are highlighted, which are shown as enlargements in the right panel. The adjacent histograms indicate the signal intensity profiles of NS5A and cholesterol along the respective arrow. Bars, 10 μm. (B) Quantification of time-dependent changes of cholesterol distribution patterns, classified into the following groups: (i) cholesterol mainly present at the plasma membrane, (ii) cholesterol mainly distributed in a vesicular pattern, and (iii) cholesterol accumulating in a web-like diffuse perinuclear structure. Results for at least two independent experiments are shown ( n , total number of cells per condition). (C) Quantification of the degrees of colocalization (Manders overlap coefficients) of NS5A and filipin at 24, 48, and 96 h postinfection. Results for two independent experiments are shown; the total number of cells per condition was ≥36. (D) Quantification of the filipin signals at 24, 48, and 72 h postinfection. The integrated fluorescence densities of the filipin signals in NS5A-positive or -negative (no virus) cells were quantified and are given in arbitrary units (AU). Results of two representative experiments are shown. The number of cells per condition was ≥11. ***, P

    Techniques Used: Infection, Fluorescence

    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

    31) Product Images from "SPANosomes as delivery vehicles for small interfering RNA (siRNA)"

    Article Title: SPANosomes as delivery vehicles for small interfering RNA (siRNA)

    Journal: Molecular Pharmaceutics

    doi: 10.1021/mp200426h

    Mechanism of SP and LF mediated siRNA transfection MDA-MB-231 cells were pre-incubated with or without an inhibitor of clathrin-mediated endocytosis (sucrose, 0.4 M), macropinocytosis (LY29004, 50 µM), and caveolae-mediated endocytosis (filipin III, 5 µg/ml) for 1 h at 37 °C. These cells were then transfected with 100 nM SP or LF/FAM-siRNA complexes in the absence or presence of the inhibitors at the same concentration for 1 h. After transfection, the cells were washed 3 times with PBS, fixed and analyzed by flow cytometry. MFI data was normalized to the cells treated without any inhibitor (100% relative MFI).
    Figure Legend Snippet: Mechanism of SP and LF mediated siRNA transfection MDA-MB-231 cells were pre-incubated with or without an inhibitor of clathrin-mediated endocytosis (sucrose, 0.4 M), macropinocytosis (LY29004, 50 µM), and caveolae-mediated endocytosis (filipin III, 5 µg/ml) for 1 h at 37 °C. These cells were then transfected with 100 nM SP or LF/FAM-siRNA complexes in the absence or presence of the inhibitors at the same concentration for 1 h. After transfection, the cells were washed 3 times with PBS, fixed and analyzed by flow cytometry. MFI data was normalized to the cells treated without any inhibitor (100% relative MFI).

    Techniques Used: Transfection, Multiple Displacement Amplification, Incubation, Concentration Assay, Flow Cytometry, Cytometry

    32) Product Images from "Exosome Uptake Depends on ERK1/2-Heat Shock Protein 27 Signaling and Lipid Raft-mediated Endocytosis Negatively Regulated by Caveolin-1 *"

    Article Title: Exosome Uptake Depends on ERK1/2-Heat Shock Protein 27 Signaling and Lipid Raft-mediated Endocytosis Negatively Regulated by Caveolin-1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.445403

    Endocytic uptake of exosomes requires intact lipid membrane rafts. A , confocal microscopy analysis shows no colocalization of Tfn ( turquoise , upper panel ) or AcLDL ( red , lower panel ) with exosomes ( red / turquoise ) at 30 min. Scale bars, 15 μm. B , knockdown validation of clathrin heavy chain using siRNA against clathrin ( si Clath ) as compared with negative control sequence ( si NT ) and normalized to α-tubulin ( C ). D , flow cytometry analysis of exosome uptake shows no difference in si Clath as compared with si NT-transfected cells. E , confocal microscopy analysis shows colocalization of CtxB (turquoise) and exosomes ( red ) at 30 min of uptake in HUVECs ( upper panel ) and U87 MG cells ( lower panel ). Scale bars, 15 μm. F , confocal microscopy analysis shows limited colocalization of Dx10 ( turquoise ) and exosomes ( red ) at 30 min of uptake in HUVECs. Scale bars, 15 μm. G , weighted colocalization coefficients display 20% (mean value) colocalization of CtxB and exosomes, and ∼15% for exosomes and 10 kDa dextran (Dx10). Colocalization coefficients were calculated (Dx10/exosomes, n = 23 cells; CtxB/exosomes, n = 22 cells) using Zeiss Zen software. *, p = 0.0182. All images were captured using a C-Apochromat 63X/1.20W korr M27 objective using laser gain of 6.0% in both lasers. H , macropinocytosis inhibitor amiloride (100 μ m ) decreases Dx10uptake (*, p = 0.01) while Tfn uptake is less affected. I , amiloride has no significant effect on exosome uptake at a wide range of concentrations. J and K , cholesterol-depleting drug MβCD dose-dependently inhibits exosome uptake. J , HUVECs ( p values, *, 0.005, **, 0.0023, ***, 0.0008); K , U87 MG cells (*, p = 0.068). L , uptake of Tfn is not affected by MβCD (2.5 m m ), while CtxB and exosome uptake are reduced, suggesting specific inhibition of lipid raft-dependent uptake ( p values, *, 0.0006, **, 0.02). M , simvastatin dose-dependently inhibits exosome uptake ( p values, *, 0.014, **, 0.011). N , sequestration of lipid rafts by filipin III substantially inhibits exosome uptake ( left panel ) while Dx10 ( middle panel ) and Tfn ( right panel ) uptake are less affected (*, p = 0.001). Presented graphs show mean fluorescent values (10,000 events/sample) of one of three representative experiments ( n = 3) with similar results ± S.D.
    Figure Legend Snippet: Endocytic uptake of exosomes requires intact lipid membrane rafts. A , confocal microscopy analysis shows no colocalization of Tfn ( turquoise , upper panel ) or AcLDL ( red , lower panel ) with exosomes ( red / turquoise ) at 30 min. Scale bars, 15 μm. B , knockdown validation of clathrin heavy chain using siRNA against clathrin ( si Clath ) as compared with negative control sequence ( si NT ) and normalized to α-tubulin ( C ). D , flow cytometry analysis of exosome uptake shows no difference in si Clath as compared with si NT-transfected cells. E , confocal microscopy analysis shows colocalization of CtxB (turquoise) and exosomes ( red ) at 30 min of uptake in HUVECs ( upper panel ) and U87 MG cells ( lower panel ). Scale bars, 15 μm. F , confocal microscopy analysis shows limited colocalization of Dx10 ( turquoise ) and exosomes ( red ) at 30 min of uptake in HUVECs. Scale bars, 15 μm. G , weighted colocalization coefficients display 20% (mean value) colocalization of CtxB and exosomes, and ∼15% for exosomes and 10 kDa dextran (Dx10). Colocalization coefficients were calculated (Dx10/exosomes, n = 23 cells; CtxB/exosomes, n = 22 cells) using Zeiss Zen software. *, p = 0.0182. All images were captured using a C-Apochromat 63X/1.20W korr M27 objective using laser gain of 6.0% in both lasers. H , macropinocytosis inhibitor amiloride (100 μ m ) decreases Dx10uptake (*, p = 0.01) while Tfn uptake is less affected. I , amiloride has no significant effect on exosome uptake at a wide range of concentrations. J and K , cholesterol-depleting drug MβCD dose-dependently inhibits exosome uptake. J , HUVECs ( p values, *, 0.005, **, 0.0023, ***, 0.0008); K , U87 MG cells (*, p = 0.068). L , uptake of Tfn is not affected by MβCD (2.5 m m ), while CtxB and exosome uptake are reduced, suggesting specific inhibition of lipid raft-dependent uptake ( p values, *, 0.0006, **, 0.02). M , simvastatin dose-dependently inhibits exosome uptake ( p values, *, 0.014, **, 0.011). N , sequestration of lipid rafts by filipin III substantially inhibits exosome uptake ( left panel ) while Dx10 ( middle panel ) and Tfn ( right panel ) uptake are less affected (*, p = 0.001). Presented graphs show mean fluorescent values (10,000 events/sample) of one of three representative experiments ( n = 3) with similar results ± S.D.

    Techniques Used: Confocal Microscopy, Negative Control, Sequencing, Flow Cytometry, Cytometry, Transfection, Software, Inhibition

    33) Product Images from "Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor"

    Article Title: Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2011.62

    Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p
    Figure Legend Snippet: Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Techniques Used: Activation Assay

    34) Product Images from "Surface Characteristics of Nanoparticles Determine Their Intracellular Fate in and Processing by Human Blood-Brain Barrier Endothelial Cells In Vitro"

    Article Title: Surface Characteristics of Nanoparticles Determine Their Intracellular Fate in and Processing by Human Blood-Brain Barrier Endothelial Cells In Vitro

    Journal: Molecular Therapy

    doi: 10.1038/mt.2010.236

    Effect of metabolic inhibitors of endocytosis on the uptake of nanoparticles. hCMEC/D3 cells were treated with filipin III (FIII; 1 µg/ml), genistein (Gen; 30 µg/ml), dimethylamiloride (DMA; 40 µmol/l),
    Figure Legend Snippet: Effect of metabolic inhibitors of endocytosis on the uptake of nanoparticles. hCMEC/D3 cells were treated with filipin III (FIII; 1 µg/ml), genistein (Gen; 30 µg/ml), dimethylamiloride (DMA; 40 µmol/l),

    Techniques Used:

    35) Product Images from "Structural and Bioactivity Characterization of Filipin Derivatives from Engineered Streptomyces filipinensis Strains Reveals Clues for Reduced Haemolytic Action"

    Article Title: Structural and Bioactivity Characterization of Filipin Derivatives from Engineered Streptomyces filipinensis Strains Reveals Clues for Reduced Haemolytic Action

    Journal: Antibiotics

    doi: 10.3390/antibiotics9070413

    Minimum inhibitory concentrations (MICs) of filipin III intermediates. Data are the average of three determinations from independent experiments. MICs of amphotericin B were determined under identical conditions. Vertical bars indicate the standard deviation (SD) values. All compounds were fungicidal at the MIC.
    Figure Legend Snippet: Minimum inhibitory concentrations (MICs) of filipin III intermediates. Data are the average of three determinations from independent experiments. MICs of amphotericin B were determined under identical conditions. Vertical bars indicate the standard deviation (SD) values. All compounds were fungicidal at the MIC.

    Techniques Used: Standard Deviation

    Haemolytic activity of filipin III and its intermediates. Haemolytic activity of pimaricin and amphotericin B was determined under identical conditions for comparison. Haemolytic activity caused by pure water was considered as 100%. Data are the average of three determinations from independent experiments. Vertical bars indicate the standard deviation values.
    Figure Legend Snippet: Haemolytic activity of filipin III and its intermediates. Haemolytic activity of pimaricin and amphotericin B was determined under identical conditions for comparison. Haemolytic activity caused by pure water was considered as 100%. Data are the average of three determinations from independent experiments. Vertical bars indicate the standard deviation values.

    Techniques Used: Activity Assay, Standard Deviation

    Chemical structures of filipin III and its precursors. COSY (H,H-correlation spectroscopy) and heteronuclear single quantum coherence (HSQC)-TOCSY correlations, as well as key heteronuclear multiple-bond correlation (HMBC) correlations for filipin I (1) and filipin II (2) are included.
    Figure Legend Snippet: Chemical structures of filipin III and its precursors. COSY (H,H-correlation spectroscopy) and heteronuclear single quantum coherence (HSQC)-TOCSY correlations, as well as key heteronuclear multiple-bond correlation (HMBC) correlations for filipin I (1) and filipin II (2) are included.

    Techniques Used: Spectroscopy

    36) Product Images from "Actin Depolymerization Disrupts Tight Junctions via Caveolae-mediated Endocytosis V⃞"

    Article Title: Actin Depolymerization Disrupts Tight Junctions via Caveolae-mediated Endocytosis V⃞

    Journal:

    doi: 10.1091/mbc.E04-12-1089

    Caveolae-mediated endocytosis is required for LatA-induced barrier dysfunction and occludin internalization. (A) Treatment of monolayers with MBCD or filipin III caused a modest reduction in TER relative to control monolayers without MBCD or filipin III
    Figure Legend Snippet: Caveolae-mediated endocytosis is required for LatA-induced barrier dysfunction and occludin internalization. (A) Treatment of monolayers with MBCD or filipin III caused a modest reduction in TER relative to control monolayers without MBCD or filipin III

    Techniques Used:

    37) 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 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

    38) Product Images from "Extracellular matrix promotes clathrin-dependent endocytosis of prolactin and STAT5 activation in differentiating mammary epithelial cells"

    Article Title: Extracellular matrix promotes clathrin-dependent endocytosis of prolactin and STAT5 activation in differentiating mammary epithelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04783-6

    Inhibition of endocytosis suppresses STAT5 activation in MECs. ( A ) Schematic of 2D lactational differentiation model. ( B ) Eph4 cells were seeded onto plastic and LrBM added to the differentiation medium as appropriate. After 24 hours, cells were stimulated with Prl (3 μg/ml) as indicated for 15 mins before lysis. Samples were analysed by SDS-PAGE/western blotting with phospho-Y 694 STAT5, total STAT5a or tubulin specific antibodies, and quantification of Odyssey scanned fluorescent images performed using ImageJ.( C – E ): Eph4 cells as in ( A ) were treated with Dyngo4 (60 µM), Pitstop II (18 µM), Filipin III (8 µM) or DMSO as indicated prior to Prl stimulation, and analysed as in ( A ). Western blots are representative of, and graphs show normalised data from at least 3 independent experiments. *p
    Figure Legend Snippet: Inhibition of endocytosis suppresses STAT5 activation in MECs. ( A ) Schematic of 2D lactational differentiation model. ( B ) Eph4 cells were seeded onto plastic and LrBM added to the differentiation medium as appropriate. After 24 hours, cells were stimulated with Prl (3 μg/ml) as indicated for 15 mins before lysis. Samples were analysed by SDS-PAGE/western blotting with phospho-Y 694 STAT5, total STAT5a or tubulin specific antibodies, and quantification of Odyssey scanned fluorescent images performed using ImageJ.( C – E ): Eph4 cells as in ( A ) were treated with Dyngo4 (60 µM), Pitstop II (18 µM), Filipin III (8 µM) or DMSO as indicated prior to Prl stimulation, and analysed as in ( A ). Western blots are representative of, and graphs show normalised data from at least 3 independent experiments. *p

    Techniques Used: Inhibition, Activation Assay, Lysis, SDS Page, Western Blot

    39) Product Images from "Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1"

    Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1056-1

    LAMP-1 is essential for cyclodextrin-mediated cholesterol export from lysosomes. Verification of HeLa cells either overexpressing LAMP-1 (HeLa-LE) or stably carrying shRNA for LAMP-1 (Hela LAMP-1 knockdown or HeLa-kd) by western blot ( a ). HeLa-LE shows significantly increased levels of LAMP-1, while HeLa-Lkd shows decreased levels of LAMP-1 compared to the HeLa cells transfected with a control plasmid. Control or transfected HeLa cells were treated with U18666A (5 μg/ml) for 48–72 h, and metabolic activity ( b ) and LDH activity ( c ) were measured. Data are mean ± S.E.M. and representative of three experiments. None of transfected cell lines showed toxicity after three days of treatment with U18666A. HeLa cells lines were analyzed for cholesterol accumulation by filipin imaging ( d ). U18666A-induced cholesterol accumulation in control cells; however, LAMP-1 overexpression (HeLa-LE) suppressed U18666A-induced cholesterol accumulation. LAMP-1 knockdown (HeLa-Lkd) did not show any protection against cholesterol accumulation and showed higher accumulation of cholesterol compared to control. Microscope images are a representative of at least three random fields of three experimental replicates. Scale bar = 50 μm
    Figure Legend Snippet: LAMP-1 is essential for cyclodextrin-mediated cholesterol export from lysosomes. Verification of HeLa cells either overexpressing LAMP-1 (HeLa-LE) or stably carrying shRNA for LAMP-1 (Hela LAMP-1 knockdown or HeLa-kd) by western blot ( a ). HeLa-LE shows significantly increased levels of LAMP-1, while HeLa-Lkd shows decreased levels of LAMP-1 compared to the HeLa cells transfected with a control plasmid. Control or transfected HeLa cells were treated with U18666A (5 μg/ml) for 48–72 h, and metabolic activity ( b ) and LDH activity ( c ) were measured. Data are mean ± S.E.M. and representative of three experiments. None of transfected cell lines showed toxicity after three days of treatment with U18666A. HeLa cells lines were analyzed for cholesterol accumulation by filipin imaging ( d ). U18666A-induced cholesterol accumulation in control cells; however, LAMP-1 overexpression (HeLa-LE) suppressed U18666A-induced cholesterol accumulation. LAMP-1 knockdown (HeLa-Lkd) did not show any protection against cholesterol accumulation and showed higher accumulation of cholesterol compared to control. Microscope images are a representative of at least three random fields of three experimental replicates. Scale bar = 50 μm

    Techniques Used: Stable Transfection, shRNA, Western Blot, Transfection, Plasmid Preparation, Activity Assay, Imaging, Over Expression, Microscopy

    40) Product Images from "Simvastatin Induced Neurite Outgrowth Unveils Role of Cell Surface Cholesterol and Acetyl CoA Carboxylase in SH-SY5Y Cells"

    Article Title: Simvastatin Induced Neurite Outgrowth Unveils Role of Cell Surface Cholesterol and Acetyl CoA Carboxylase in SH-SY5Y Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074547

    Retention of CSC in plasma membrane determines SIM induced neuritogenesis in SH-SY5Y cells. A ) Confocal image of SH-SY5Y cells at 60 X magnification showing fluorescence of cholesterol binding probe i.e. Filipin III in control (a); and SIM treated cells for 2 hrs (b), 6 hrs (c) and 12 hrs (d). The fluorescence (shown as blue fluorescence) was observed not only in plasma membrane but also in neurites. The neurite has been represented by *. N = 25-30 per condition from 3 separate cultures (n = 3). Scale Bar = 10 µm. B ) Thin layer chromatography showing total cellular cholesterol content at 2 hrs, 6 hrs and 12 hrs after SIM treatment. A significant ( p
    Figure Legend Snippet: Retention of CSC in plasma membrane determines SIM induced neuritogenesis in SH-SY5Y cells. A ) Confocal image of SH-SY5Y cells at 60 X magnification showing fluorescence of cholesterol binding probe i.e. Filipin III in control (a); and SIM treated cells for 2 hrs (b), 6 hrs (c) and 12 hrs (d). The fluorescence (shown as blue fluorescence) was observed not only in plasma membrane but also in neurites. The neurite has been represented by *. N = 25-30 per condition from 3 separate cultures (n = 3). Scale Bar = 10 µm. B ) Thin layer chromatography showing total cellular cholesterol content at 2 hrs, 6 hrs and 12 hrs after SIM treatment. A significant ( p

    Techniques Used: Fluorescence, Binding Assay, Thin Layer Chromatography

    Related Articles

    Flow Cytometry:

    Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol
    Article Snippet: .. Imaging flow cytometry was performed on 1 million cells per sample, which were washed with PBS, fixed with 3.7% PFA for 20 min at RT, and stained with WGA Alexa Fluor 680 (Thermo Fisher) for 10 min, 0.05 mg/mL Filipin (Sigma) for 2 h, or both stains and were run through the ImageStream X Flow Cytometer (Amnis Corp.). .. Analysis was performed using the ISX software to determine colocalization and intensity of WGA and filipin at the plasma membrane.

    Whole Genome Amplification:

    Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol
    Article Snippet: .. Imaging flow cytometry was performed on 1 million cells per sample, which were washed with PBS, fixed with 3.7% PFA for 20 min at RT, and stained with WGA Alexa Fluor 680 (Thermo Fisher) for 10 min, 0.05 mg/mL Filipin (Sigma) for 2 h, or both stains and were run through the ImageStream X Flow Cytometer (Amnis Corp.). .. Analysis was performed using the ISX software to determine colocalization and intensity of WGA and filipin at the plasma membrane.

    Cytometry:

    Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol
    Article Snippet: .. Imaging flow cytometry was performed on 1 million cells per sample, which were washed with PBS, fixed with 3.7% PFA for 20 min at RT, and stained with WGA Alexa Fluor 680 (Thermo Fisher) for 10 min, 0.05 mg/mL Filipin (Sigma) for 2 h, or both stains and were run through the ImageStream X Flow Cytometer (Amnis Corp.). .. Analysis was performed using the ISX software to determine colocalization and intensity of WGA and filipin at the plasma membrane.

    Microscopy:

    Article Title: INCREASED MEMBRANE CHOLESTEROL MIGHT RENDER MATURE HIPPOCAMPAL NEURONS MORE SUSCEPTIBLE TO BETA-AMYLOID-INDUCED CALPAIN ACTIVATION AND TAU TOXICITY
    Article Snippet: .. Hippocampal neurons were cultured for 7 or 21 days on coverslips, after which they were fixed in 4% PFA in PBS containing 0.12 mM sucrose for 15 min and labeled with 300 µg/ml filipin (Sigma) in PBS for 90 min. After washing with PBS, the cells were fixed for a second time in PFA for 20 min and mounted on a microscope slide. .. Filipin fluorescence was analyzed 1 d after staining by taking images at 40X magnification and 5 second exposures using MetaMorph Image Analysis software (Universal Imaging Corporation) and a Photometrics Cool Snap HQ2 camera coupled to a fluorescent microscope (Nikon Diaphot).

    Labeling:

    Article Title: INCREASED MEMBRANE CHOLESTEROL MIGHT RENDER MATURE HIPPOCAMPAL NEURONS MORE SUSCEPTIBLE TO BETA-AMYLOID-INDUCED CALPAIN ACTIVATION AND TAU TOXICITY
    Article Snippet: .. Hippocampal neurons were cultured for 7 or 21 days on coverslips, after which they were fixed in 4% PFA in PBS containing 0.12 mM sucrose for 15 min and labeled with 300 µg/ml filipin (Sigma) in PBS for 90 min. After washing with PBS, the cells were fixed for a second time in PFA for 20 min and mounted on a microscope slide. .. Filipin fluorescence was analyzed 1 d after staining by taking images at 40X magnification and 5 second exposures using MetaMorph Image Analysis software (Universal Imaging Corporation) and a Photometrics Cool Snap HQ2 camera coupled to a fluorescent microscope (Nikon Diaphot).

    other:

    Article Title: S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *
    Article Snippet: OA, caprylate, stearate, GSH, 1,4-dithiothreitol (DTT), and filipin III were purchased from Sigma-Aldrich.

    Article Title: Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway ▿
    Article Snippet: Additional reagents used in this study were obtained from the following sources: fluorochrome-conjugated secondary antibodies and the DNA dye Hoechst 33342 were obtained from Molecular Probes/Invitrogen, Eugene, OR; nocodazole and brefeldin A were obtained from EMD Chemicals, Gibbstown, NJ; and U18666A, hydrochloride, and filipin were obtained from Sigma, St. Louis, MO.

    Article Title: Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium
    Article Snippet: Reagents 17β-Estradiol (E2 ), ICI 182,780 (ICI; an ER antagonist), tamoxifen, clomiphene citrate, U18666A, and filipin were purchased from Sigma-Aldrich (Sigma, US).

    Cell Culture:

    Article Title: INCREASED MEMBRANE CHOLESTEROL MIGHT RENDER MATURE HIPPOCAMPAL NEURONS MORE SUSCEPTIBLE TO BETA-AMYLOID-INDUCED CALPAIN ACTIVATION AND TAU TOXICITY
    Article Snippet: .. Hippocampal neurons were cultured for 7 or 21 days on coverslips, after which they were fixed in 4% PFA in PBS containing 0.12 mM sucrose for 15 min and labeled with 300 µg/ml filipin (Sigma) in PBS for 90 min. After washing with PBS, the cells were fixed for a second time in PFA for 20 min and mounted on a microscope slide. .. Filipin fluorescence was analyzed 1 d after staining by taking images at 40X magnification and 5 second exposures using MetaMorph Image Analysis software (Universal Imaging Corporation) and a Photometrics Cool Snap HQ2 camera coupled to a fluorescent microscope (Nikon Diaphot).

    Staining:

    Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol
    Article Snippet: .. Imaging flow cytometry was performed on 1 million cells per sample, which were washed with PBS, fixed with 3.7% PFA for 20 min at RT, and stained with WGA Alexa Fluor 680 (Thermo Fisher) for 10 min, 0.05 mg/mL Filipin (Sigma) for 2 h, or both stains and were run through the ImageStream X Flow Cytometer (Amnis Corp.). .. Analysis was performed using the ISX software to determine colocalization and intensity of WGA and filipin at the plasma membrane.

    Imaging:

    Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol
    Article Snippet: .. Imaging flow cytometry was performed on 1 million cells per sample, which were washed with PBS, fixed with 3.7% PFA for 20 min at RT, and stained with WGA Alexa Fluor 680 (Thermo Fisher) for 10 min, 0.05 mg/mL Filipin (Sigma) for 2 h, or both stains and were run through the ImageStream X Flow Cytometer (Amnis Corp.). .. Analysis was performed using the ISX software to determine colocalization and intensity of WGA and filipin at the plasma membrane.

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  • 93
    Millipore filipin
    StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with <t>filipin</t> and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fluorescent filipin iii
    Membrane cholesterol accumulation during adipogenesis. The membrane cholesterol was stained with fresh <t>filipin</t> <t>III</t> (green) and imaged at day 0 (A) and day 7 (B), and overlaid with brightfield images. These images were recorded using a 60×/1.4 NA microscope objective. Bar = 20 μ m.
    Fluorescent Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescent filipin iii/product/Millipore
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    91
    Millipore filipin staining
    Acetyl-CoA carboxylase (ACC) inhibition redistributes free cholesterol and endosome distribution in cystic fibrosis (CF) epithelial cells. A , left : control-S9 and IB3 cells were treated with vehicle [not treated (NT)] or 10 µg/ml 5-(tetradecyloxy)-2-furoic acid (TOFA) for 24 h and assessed for cholesterol distribution by <t>filipin</t> staining. Representative images are shown from 5 separate experiments. A , right : filipin-stained images were quantified for perinuclear cholesterol accumulation and data are presented as percentage of cells with perinuclear accumulation ( n = 20 separate images for each condition, * P
    Filipin Staining, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with filipin and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P

    Journal: Journal of Lipid Research

    Article Title: StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis [S]

    doi: 10.1194/jlr.M091967

    Figure Lengend Snippet: StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with filipin and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P

    Article Snippet: Filipin was from Cayman Biochemicals; cholesterol oxidase was from Calbiochem, BODIPY 493/503 was from Fisher Scientific; cAMP, cyclosporin A, and other general chemicals were from Sigma.

    Techniques: Mouse Assay, Staining, Infection

    Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor

    doi: 10.1038/labinvest.2011.62

    Figure Lengend Snippet: Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Article Snippet: The inhibitors utilized were the lipid raft disruptors, methyl-β-cyclodextrin (0.1 mM), , and filipin III (1 mg/mL) ( , ), and the JNK inhibitor (10−7 M; SP600125; EMD Chemicals, Gibbstown, NJ) ( ).

    Techniques: Activation Assay

    Membrane cholesterol accumulation during adipogenesis. The membrane cholesterol was stained with fresh filipin III (green) and imaged at day 0 (A) and day 7 (B), and overlaid with brightfield images. These images were recorded using a 60×/1.4 NA microscope objective. Bar = 20 μ m.

    Journal: Journal of Biomechanical Engineering

    Article Title: Cholesterol-Dependent Modulation of Stem Cell Biomechanics: Application to Adipogenesis

    doi: 10.1115/1.4043253

    Figure Lengend Snippet: Membrane cholesterol accumulation during adipogenesis. The membrane cholesterol was stained with fresh filipin III (green) and imaged at day 0 (A) and day 7 (B), and overlaid with brightfield images. These images were recorded using a 60×/1.4 NA microscope objective. Bar = 20 μ m.

    Article Snippet: Fluorescent filipin III was used to stain the membrane cholesterol.

    Techniques: Staining, Microscopy

    Acetyl-CoA carboxylase (ACC) inhibition redistributes free cholesterol and endosome distribution in cystic fibrosis (CF) epithelial cells. A , left : control-S9 and IB3 cells were treated with vehicle [not treated (NT)] or 10 µg/ml 5-(tetradecyloxy)-2-furoic acid (TOFA) for 24 h and assessed for cholesterol distribution by filipin staining. Representative images are shown from 5 separate experiments. A , right : filipin-stained images were quantified for perinuclear cholesterol accumulation and data are presented as percentage of cells with perinuclear accumulation ( n = 20 separate images for each condition, * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells

    doi: 10.1152/ajplung.00369.2018

    Figure Lengend Snippet: Acetyl-CoA carboxylase (ACC) inhibition redistributes free cholesterol and endosome distribution in cystic fibrosis (CF) epithelial cells. A , left : control-S9 and IB3 cells were treated with vehicle [not treated (NT)] or 10 µg/ml 5-(tetradecyloxy)-2-furoic acid (TOFA) for 24 h and assessed for cholesterol distribution by filipin staining. Representative images are shown from 5 separate experiments. A , right : filipin-stained images were quantified for perinuclear cholesterol accumulation and data are presented as percentage of cells with perinuclear accumulation ( n = 20 separate images for each condition, * P

    Article Snippet: Filipin staining.

    Techniques: Inhibition, Staining