filipin iii  (Millipore)


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  • 95
    Name:
    Filipin III from Streptomyces filipinensis
    Description:

    Catalog Number:
    f4767
    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 from Streptomyces filipinensis

    https://www.bioz.com/result/filipin iii/product/Millipore
    Average 95 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    filipin iii - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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

    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 "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

    6) 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

    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 "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

    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 "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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    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 "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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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:

    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 "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

    28) 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

    29) 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

    30) 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

    31) Product Images from "Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism"

    Article Title: Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism

    Journal: Nature

    doi: 10.1038/nature17412

    Plasma membrane cholesterol modulates TCR clustering and immunological synapse formation a – d , Cholesterol quantification of naive and activated CD8 + T cells by filipin III staining ( a , b ), oxidation-based method ( c ) or biotinylation-based method ( d ) ( a , b , n = 60; c , d , n = 4). PM, plasma membrane. e , CD3 and CD8 surface levels of naive wild-type and CKO CD8 + T cells. f , TCR signalling of naive CD8 + T cells stimulated with 4 μg ml −1 anti-CD3/CD28. See Supplementary Fig. 1 for gel source data. g – i , STORM analysis of TCR clustering in naive and activated CD8 + T cells. g , Representative images. h , Ripley’s K -function analysis of TCR molecules. r , radius. i , The r value at the maximal L ( r ) − r value of Ripley’s K -function curves (naive, WT, n = 29, CKO, n = 22; activated, WT, n = 19, CKO, n = 17). j , k , Total internal reflection fluorescence microscopy (TIRFM) analysis of immunological synapse of CD8 + T cells on stimulatory planar lipid bilayer. j , Representative images. k , Immunological synapse area ( n = 13 cells). l – n , Parameters of TCR microcluster movements of CD8 + T cells. l , Mean square displacements (MSD). TCR microcluster movements were split into directed, confined and random movements. m , Cumulative probability distribution (CPD). n , Mean scattered plots of diffusion coefficient. TCR microclusters were from 19 WT and 20 CKO cells. o , p , Cytolytic granule polarization ( o , n = 50) and degranulation ( p , n = 3) of OT-I CTLs. Data are representative of two ( a – c , j – n , p ) or three ( f – i , o ) independent experiments, and were analysed by unpaired t -test ( c , d , p ), Mann–Whitney test ( b , i , n , o ), two-way ANOVA ( k , l ) or Kolmogorov–Smirnov test ( m ). Error bars denote s.e.m. * P
    Figure Legend Snippet: Plasma membrane cholesterol modulates TCR clustering and immunological synapse formation a – d , Cholesterol quantification of naive and activated CD8 + T cells by filipin III staining ( a , b ), oxidation-based method ( c ) or biotinylation-based method ( d ) ( a , b , n = 60; c , d , n = 4). PM, plasma membrane. e , CD3 and CD8 surface levels of naive wild-type and CKO CD8 + T cells. f , TCR signalling of naive CD8 + T cells stimulated with 4 μg ml −1 anti-CD3/CD28. See Supplementary Fig. 1 for gel source data. g – i , STORM analysis of TCR clustering in naive and activated CD8 + T cells. g , Representative images. h , Ripley’s K -function analysis of TCR molecules. r , radius. i , The r value at the maximal L ( r ) − r value of Ripley’s K -function curves (naive, WT, n = 29, CKO, n = 22; activated, WT, n = 19, CKO, n = 17). j , k , Total internal reflection fluorescence microscopy (TIRFM) analysis of immunological synapse of CD8 + T cells on stimulatory planar lipid bilayer. j , Representative images. k , Immunological synapse area ( n = 13 cells). l – n , Parameters of TCR microcluster movements of CD8 + T cells. l , Mean square displacements (MSD). TCR microcluster movements were split into directed, confined and random movements. m , Cumulative probability distribution (CPD). n , Mean scattered plots of diffusion coefficient. TCR microclusters were from 19 WT and 20 CKO cells. o , p , Cytolytic granule polarization ( o , n = 50) and degranulation ( p , n = 3) of OT-I CTLs. Data are representative of two ( a – c , j – n , p ) or three ( f – i , o ) independent experiments, and were analysed by unpaired t -test ( c , d , p ), Mann–Whitney test ( b , i , n , o ), two-way ANOVA ( k , l ) or Kolmogorov–Smirnov test ( m ). Error bars denote s.e.m. * P

    Techniques Used: Staining, Fluorescence, Microscopy, Diffusion-based Assay, MANN-WHITNEY

    Reprogramming of cellular cholesterol metabolism in activated CD8 + T cells a , Filipin III staining (left) and quantification (right) of cellular cholesterol of naive and activated CD8 + T cells stimulated by 5 μg ml −1 plate-bound anti-CD3/CD28 antibodies for 12 h ( n = 60). b , Total cellular, plasma membrane and intracellular cholesterol quantified using the cholesterol oxidation-based method ( n = 4). c , Relative plasma membrane cholesterol quantified using the biotinylation-based method ( n = 4). d – f , Transcriptional levels of key genes encoding molecules involved in cholesterol synthesis, transport and efflux ( n = 3). CD8 + T cells were stimulated with 5 μg ml −1 plate-bound anti-CD3/CD28 antibodies. Results and statistical analysis are relative to quiescent CD8 + T cells. mRNA levels of cholesterol biosynthesis genes, including Srebp1 (also known as Srebf1 ), Srebp2 ( Srebf2 ), Hmgcr , Hmgcs , Fasn , Acaca and Sqle , were upregulated in activated CD8 + T cells. Ldlr , which encodes the LDL receptor, a major receptor for cholesterol transport, was upregulated in activated CD8 + T cells, whereas, Idol (also known as Mylip ), which encodes IDOL, an inducible degrader of the LDL receptor, was downregulated. Cholesterol efflux genes, including Abca1 and Abcg1 , were all downregulated in activated CD8 + T cells. g – i , Cytokine/granule productions of CD8 + T cells after modulation of cholesterol metabolic pathways ( n = 3). Naive CD8 + T cells were pretreated for 6 h with vehicle (DMSO), lovastatin (to inhibit cholesterol biosynthesis) or U18666A (a cholesterol transport inhibitor with pleotropic effects), respectively. Cells were then stimulated with 5 μg ml −1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h before intracellular staining. Representative flow cytometric profiles shown in g . Data are representative of two independent experiments, and were analysed by Mann–Whitney test ( a ) or unpaired t -test ( b – f , h , i ). Error bars denote s.e.m; * P
    Figure Legend Snippet: Reprogramming of cellular cholesterol metabolism in activated CD8 + T cells a , Filipin III staining (left) and quantification (right) of cellular cholesterol of naive and activated CD8 + T cells stimulated by 5 μg ml −1 plate-bound anti-CD3/CD28 antibodies for 12 h ( n = 60). b , Total cellular, plasma membrane and intracellular cholesterol quantified using the cholesterol oxidation-based method ( n = 4). c , Relative plasma membrane cholesterol quantified using the biotinylation-based method ( n = 4). d – f , Transcriptional levels of key genes encoding molecules involved in cholesterol synthesis, transport and efflux ( n = 3). CD8 + T cells were stimulated with 5 μg ml −1 plate-bound anti-CD3/CD28 antibodies. Results and statistical analysis are relative to quiescent CD8 + T cells. mRNA levels of cholesterol biosynthesis genes, including Srebp1 (also known as Srebf1 ), Srebp2 ( Srebf2 ), Hmgcr , Hmgcs , Fasn , Acaca and Sqle , were upregulated in activated CD8 + T cells. Ldlr , which encodes the LDL receptor, a major receptor for cholesterol transport, was upregulated in activated CD8 + T cells, whereas, Idol (also known as Mylip ), which encodes IDOL, an inducible degrader of the LDL receptor, was downregulated. Cholesterol efflux genes, including Abca1 and Abcg1 , were all downregulated in activated CD8 + T cells. g – i , Cytokine/granule productions of CD8 + T cells after modulation of cholesterol metabolic pathways ( n = 3). Naive CD8 + T cells were pretreated for 6 h with vehicle (DMSO), lovastatin (to inhibit cholesterol biosynthesis) or U18666A (a cholesterol transport inhibitor with pleotropic effects), respectively. Cells were then stimulated with 5 μg ml −1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h before intracellular staining. Representative flow cytometric profiles shown in g . Data are representative of two independent experiments, and were analysed by Mann–Whitney test ( a ) or unpaired t -test ( b – f , h , i ). Error bars denote s.e.m; * P

    Techniques Used: Staining, Flow Cytometry, MANN-WHITNEY

    Avasimibe treatment leads to enhanced TCR clustering and signalling, as well as more efficient formation of immunological synapse a , Cytokine/granule productions of CD8 + T cells after avasimibe treatment ( n = 3). The naive cells were pretreated for 6 h with avasimibe or vehicle (DMSO) and then stimulated by 5 μg ml −1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. Data were analysed by t -test. b, CTL cytotoxicity after avasimibe treatment measured by the LDH assay ( n = 3). OT-I CTLs were pretreated with avasimibe or vehicle for 6 h and then incubated with EL-4 cells pulsed with OVA 257–264 peptide for 4 h. Data were analysed by t -test. c , An MTS-based cell viability assay was performed to assess the toxicity of avasimibe to B16F10 cells ( n = 6). Data were analysed by one-way ANOVA, and no significant difference was observed. d , e , Filipin III staining to analyse cellular cholesterol distribution in naive CD8 + T cells treated with avasimibe or vehicle. d , Representative images. e , Data were analysed by Mann–Whitney test. 0, n = 217; 0.5, n = 139; 1, n = 133. f – h , Super-resolution STORM images of TCR in naive CD8 + T cells treated with avasimibe or vehicle. f , Representative images. g, Ripley’s K -function was used to analyse TCR molecules distribution. h , The r value at the maximal L ( r ) − r value of Ripley’s K -function curves, and data were analysed by Mann–Whitney test. 0, n = 41; 0.5, n = 60; 1, n = 66. i , Representative TIRFM images of immunological synapses of CD8 + T cells treated with avasimibe (1 μM) or vehicle for 6 h. Cells were stimulated by PLB-bound anti-CD3 for the indicated time and fixed by 4% PFA before imaging. j , Areas of the immunological synapses ( n > 60 cells). The formation and contraction of the immunological synapses of CD8 + T cells treated with avasimibe were more rapid than those treated with vehicle. Data were analysed by two-way ANOVA. k , TCR proximal and downstream signalling was assessed using immunoblotting of protein phosphorylation. OT-I CTLs were treated with 1 μM avasimibe or vehicle for 6 h and then stimulated with 2 μg ml −1 soluble anti-CD3 and anti-CD28 for the indicated time. See Supplementary Fig. 1 for gel source data. Error bars denote s.e.m; * P
    Figure Legend Snippet: Avasimibe treatment leads to enhanced TCR clustering and signalling, as well as more efficient formation of immunological synapse a , Cytokine/granule productions of CD8 + T cells after avasimibe treatment ( n = 3). The naive cells were pretreated for 6 h with avasimibe or vehicle (DMSO) and then stimulated by 5 μg ml −1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. Data were analysed by t -test. b, CTL cytotoxicity after avasimibe treatment measured by the LDH assay ( n = 3). OT-I CTLs were pretreated with avasimibe or vehicle for 6 h and then incubated with EL-4 cells pulsed with OVA 257–264 peptide for 4 h. Data were analysed by t -test. c , An MTS-based cell viability assay was performed to assess the toxicity of avasimibe to B16F10 cells ( n = 6). Data were analysed by one-way ANOVA, and no significant difference was observed. d , e , Filipin III staining to analyse cellular cholesterol distribution in naive CD8 + T cells treated with avasimibe or vehicle. d , Representative images. e , Data were analysed by Mann–Whitney test. 0, n = 217; 0.5, n = 139; 1, n = 133. f – h , Super-resolution STORM images of TCR in naive CD8 + T cells treated with avasimibe or vehicle. f , Representative images. g, Ripley’s K -function was used to analyse TCR molecules distribution. h , The r value at the maximal L ( r ) − r value of Ripley’s K -function curves, and data were analysed by Mann–Whitney test. 0, n = 41; 0.5, n = 60; 1, n = 66. i , Representative TIRFM images of immunological synapses of CD8 + T cells treated with avasimibe (1 μM) or vehicle for 6 h. Cells were stimulated by PLB-bound anti-CD3 for the indicated time and fixed by 4% PFA before imaging. j , Areas of the immunological synapses ( n > 60 cells). The formation and contraction of the immunological synapses of CD8 + T cells treated with avasimibe were more rapid than those treated with vehicle. Data were analysed by two-way ANOVA. k , TCR proximal and downstream signalling was assessed using immunoblotting of protein phosphorylation. OT-I CTLs were treated with 1 μM avasimibe or vehicle for 6 h and then stimulated with 2 μg ml −1 soluble anti-CD3 and anti-CD28 for the indicated time. See Supplementary Fig. 1 for gel source data. Error bars denote s.e.m; * P

    Techniques Used: CTL Assay, Lactate Dehydrogenase Assay, Incubation, Viability Assay, Staining, MANN-WHITNEY, Imaging

    ACAT1 deficiency does not result in significant change of CD4 + T-cell function a , b , Cytokine productions of CD4 + T cells ( n = 3). Cells were stimulated with 5 μg ml −1 plate-bound anti-CD3 and anti-CD28 antibodies for 12 h. Representative flow cytometric profiles are shown in a. c – e , Relative transcription levels of Acat1 and Acat2 in naive CD4 + and CD8 + T cells freshly isolated from C57BL/6 mice ( n = 3). Acat1 transcription level was significantly higher than Acat2 in CD4 + T cells. Acat1 transcription levels were comparable between CD4 + and CD8 + T cells, whereas the Acat2 transcription level in CD4 + T cells was significantly higher than that in CD8 + T cells. Acat2 transcription level in CD4 + T cells was set as 1 in c . Acat1 and Acat2 transcription levels in CD8 + T cells were set as 1 in d and e. f , Filipin III staining to analyse cellular cholesterol distribution in naive and activated CD4 + T cells from wild-type and CKO mice. Data were analysed by unpaired t -test ( b – e ) or Mann–Whitney test ( f ). Error bars denote s.e.m; * P
    Figure Legend Snippet: ACAT1 deficiency does not result in significant change of CD4 + T-cell function a , b , Cytokine productions of CD4 + T cells ( n = 3). Cells were stimulated with 5 μg ml −1 plate-bound anti-CD3 and anti-CD28 antibodies for 12 h. Representative flow cytometric profiles are shown in a. c – e , Relative transcription levels of Acat1 and Acat2 in naive CD4 + and CD8 + T cells freshly isolated from C57BL/6 mice ( n = 3). Acat1 transcription level was significantly higher than Acat2 in CD4 + T cells. Acat1 transcription levels were comparable between CD4 + and CD8 + T cells, whereas the Acat2 transcription level in CD4 + T cells was significantly higher than that in CD8 + T cells. Acat2 transcription level in CD4 + T cells was set as 1 in c . Acat1 and Acat2 transcription levels in CD8 + T cells were set as 1 in d and e. f , Filipin III staining to analyse cellular cholesterol distribution in naive and activated CD4 + T cells from wild-type and CKO mice. Data were analysed by unpaired t -test ( b – e ) or Mann–Whitney test ( f ). Error bars denote s.e.m; * P

    Techniques Used: Cell Function Assay, Flow Cytometry, Isolation, Mouse Assay, Staining, MANN-WHITNEY

    32) 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:

    33) 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

    34) Product Images from "Neuronal cholesterol metabolism increases dendritic outgrowth and synaptic markers via a concerted action of GGTase-I and Trk"

    Article Title: Neuronal cholesterol metabolism increases dendritic outgrowth and synaptic markers via a concerted action of GGTase-I and Trk

    Journal: Scientific Reports

    doi: 10.1038/srep30928

    Cholesterol reduction is necessary for CYP46A1 induced dendritic outgrowth. ( A,B ) Primary cultures of rat cortical 4DIV neurons were transfected with Control or hCYP vector and maintained for 48 hours. 24 hours after transfection cells were incubated with 10 μM cholesterol (Chol:MBCD). Neurons were stained with filipin III (free cholesterol staining) and immunostained for MAP2 and FLAG. Scale bar: 20 μm. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test (* p
    Figure Legend Snippet: Cholesterol reduction is necessary for CYP46A1 induced dendritic outgrowth. ( A,B ) Primary cultures of rat cortical 4DIV neurons were transfected with Control or hCYP vector and maintained for 48 hours. 24 hours after transfection cells were incubated with 10 μM cholesterol (Chol:MBCD). Neurons were stained with filipin III (free cholesterol staining) and immunostained for MAP2 and FLAG. Scale bar: 20 μm. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test (* p

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Staining

    Cholesterol reduction is a necessary trigger for the CYP46A1-dependent increase in dendritic protrusion density and p-Trk levels. ( A,B ) Primary cultures of rat cortical neurons 19 DIV were transduced with AdControl or AdCYP adenovirus and maintained for 48 hours. 24 hours after transduction cells were incubated with 10 μM Chol:MBCD. Neurons were stained with filipin III (blue) and immunstained with p-Trk (red). Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test (* p
    Figure Legend Snippet: Cholesterol reduction is a necessary trigger for the CYP46A1-dependent increase in dendritic protrusion density and p-Trk levels. ( A,B ) Primary cultures of rat cortical neurons 19 DIV were transduced with AdControl or AdCYP adenovirus and maintained for 48 hours. 24 hours after transduction cells were incubated with 10 μM Chol:MBCD. Neurons were stained with filipin III (blue) and immunstained with p-Trk (red). Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test (* p

    Techniques Used: Transduction, Incubation, Staining

    35) 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

    36) 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

    37) Product Images from "B Cell Activation by Outer Membrane Vesicles--A Novel Virulence Mechanism"

    Article Title: B Cell Activation by Outer Membrane Vesicles--A Novel Virulence Mechanism

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000724

    Ca 2+ mobilization and B cell receptor clustering are induced by OMV. (A) Mobilization of [Ca 2+ ] i in human tonsillar B cells induced by Moraxella OMV. Purified Fura-2 loaded B cells were stimulated with wild-type OMV (10 µg/ml), MID-deficient OMV (10 µg/ml) or ionomycin (100 nM). Ca 2+ mobilization was analyzed as described in Materials and Methods . Each experiment was repeated with at least three different donors with comparable results. (B) Aliquots of Triton-insoluble lysates of B cells obtained before (Control) and after stimulation with formaldehyde-fixed M . catarrhalis wild type (Bacteria), OMV or treated with filipin prior to stimulation with OMV (Filipin + OMV) were fractionated on discontinuous sucrose gradients and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR2, anti-IgD or anti-TLR9 mAbs. (C) Visualization of receptor clustering in lipid rafts. B cells unstimulated (Control), wild type OMV (OMV wt) or MID-deficient OMV (OMV Δ mid ) stimulated for 30 min were fixed and stained with anti-flotillin (top) or anti-TLR2 (bottom). Alexa Fluor 594 goat anti-mouse IgG pAb were used as a secondary layer. (D) Colocalization of flotillin, TLR2, Rab5 and TLR9 with IgD (BCR) confirmed by confocal microscopy. Purified B cells were incubated with OMV for 30 min, fixed and stained with FITC-conjugated or RPE-conjugated mAbs against IgD (BCR) and FITC-conjugated anti-TLR9 mAb, anti-flotillin mAb or anti-TLR2 mAb using Alexa Fluor 594-conjugated secondary pAb. For the triple staining, stimulated B cells were incubated with rabbit anti-IgD pAb (BCR) and mouse anti-Rab5 mAb (Rab5). After several washes, B cells were incubated with Alexa Fluor 633 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG secondary mAb followed by incubation with FITC-conjugated anti-TLR9 mAb (TLR9). The lymphocytes shown are representatives of more than 90% of the cells imaged in 3 separate experiments with different donors.
    Figure Legend Snippet: Ca 2+ mobilization and B cell receptor clustering are induced by OMV. (A) Mobilization of [Ca 2+ ] i in human tonsillar B cells induced by Moraxella OMV. Purified Fura-2 loaded B cells were stimulated with wild-type OMV (10 µg/ml), MID-deficient OMV (10 µg/ml) or ionomycin (100 nM). Ca 2+ mobilization was analyzed as described in Materials and Methods . Each experiment was repeated with at least three different donors with comparable results. (B) Aliquots of Triton-insoluble lysates of B cells obtained before (Control) and after stimulation with formaldehyde-fixed M . catarrhalis wild type (Bacteria), OMV or treated with filipin prior to stimulation with OMV (Filipin + OMV) were fractionated on discontinuous sucrose gradients and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR2, anti-IgD or anti-TLR9 mAbs. (C) Visualization of receptor clustering in lipid rafts. B cells unstimulated (Control), wild type OMV (OMV wt) or MID-deficient OMV (OMV Δ mid ) stimulated for 30 min were fixed and stained with anti-flotillin (top) or anti-TLR2 (bottom). Alexa Fluor 594 goat anti-mouse IgG pAb were used as a secondary layer. (D) Colocalization of flotillin, TLR2, Rab5 and TLR9 with IgD (BCR) confirmed by confocal microscopy. Purified B cells were incubated with OMV for 30 min, fixed and stained with FITC-conjugated or RPE-conjugated mAbs against IgD (BCR) and FITC-conjugated anti-TLR9 mAb, anti-flotillin mAb or anti-TLR2 mAb using Alexa Fluor 594-conjugated secondary pAb. For the triple staining, stimulated B cells were incubated with rabbit anti-IgD pAb (BCR) and mouse anti-Rab5 mAb (Rab5). After several washes, B cells were incubated with Alexa Fluor 633 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG secondary mAb followed by incubation with FITC-conjugated anti-TLR9 mAb (TLR9). The lymphocytes shown are representatives of more than 90% of the cells imaged in 3 separate experiments with different donors.

    Techniques Used: Purification, Staining, Confocal Microscopy, Incubation

    38) 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

    39) Product Images from "The Chemokine Receptor CCR2 Mediates the Binding and Internalization of Monocyte Chemoattractant Protein-1 along Brain Microvessels"

    Article Title: The Chemokine Receptor CCR2 Mediates the Binding and Internalization of Monocyte Chemoattractant Protein-1 along Brain Microvessels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-23-09214.2001

    Effect of filipin III on internalization of MCP-1 along murine brain microvessels. To gauge whether MCP-1 internalization may be mediated by caveolae, we exposed the microvessels to the caveolae-disrupting agent filipin III before incubation with biot.-rmMCP-1 at 37°C. Contrary to nontreated controls, the microvessels pretreated with filipin III did not require permeabilization to enable the detection of labeled chemokine, suggesting that biot.-rmMCP-1 remained on the cell surface as a consequence of caveolar disruption. Scale bar, 50 μm.
    Figure Legend Snippet: Effect of filipin III on internalization of MCP-1 along murine brain microvessels. To gauge whether MCP-1 internalization may be mediated by caveolae, we exposed the microvessels to the caveolae-disrupting agent filipin III before incubation with biot.-rmMCP-1 at 37°C. Contrary to nontreated controls, the microvessels pretreated with filipin III did not require permeabilization to enable the detection of labeled chemokine, suggesting that biot.-rmMCP-1 remained on the cell surface as a consequence of caveolar disruption. Scale bar, 50 μm.

    Techniques Used: Incubation, Labeling

    Colocalization of internalized MCP-1 with caveolin-1. Microvessels were pretreated (±) with filipin III. Then the samples were exposed to biot.-rmMCP-1 at 4 or 37°C, fixed/permeabilized, and stained to reveal labeled chemokine ( green ) and caveolin-1 ( red ) localization, as described in Materials and Methods. Then confocal images were obtained at a level approximately midway through the interior of the microvascular samples, revealing the relative distribution patterns of labeled chemokine and caveolin-1. In the control sample exposed to biot.-rmMCP-1 at 37°C, caveolin-1 staining can be seen concentrated around the periphery of the microvascular segment ( arrows ), with chemokine apparently present diffusely in the cytoplasm. Areas of yellow fluorescence ( asterisks ) indicate presumed sites of biot.-rmMCP-1/caveolin-1 colocalization. In the filipin III-treated sample exposed to chemokine at 37°C, no sites of colocalization are detected readily, and biot.-rmMCP-1 appears to be confined to the membrane surface ( arrowheads ), with caveolin-1 expression heightened in some cytoplasmic locales ( arrows ). Microvessels exposed to chemokine at 4°C (±) filipin III pretreatment also fail to show any areas of biot.-rmMCP-1/caveolin-1 colocalization. These samples also do not demonstrate as strong a cytoplasmic distribution of labeled chemokine as that observed in the control sample at 37°C but seemingly manifest a more peripheral chemokine staining ( arrowheads ), possibly restricted to the membrane surface. As with the samples exposed to chemokine at 37°C, caveolin-1 distribution appears to be concentrated along the periphery of the microvessel in the control ( arrowheads ) but is dispersed more cytoplasmically in the filipin III-treated sample. Arrows , Caveolin-1; arrowheads , biot.-rmMCP-1; asterisks , biot.-rmMCP-1/caveolin-1 colocalization. Scale bar, 10 μm.
    Figure Legend Snippet: Colocalization of internalized MCP-1 with caveolin-1. Microvessels were pretreated (±) with filipin III. Then the samples were exposed to biot.-rmMCP-1 at 4 or 37°C, fixed/permeabilized, and stained to reveal labeled chemokine ( green ) and caveolin-1 ( red ) localization, as described in Materials and Methods. Then confocal images were obtained at a level approximately midway through the interior of the microvascular samples, revealing the relative distribution patterns of labeled chemokine and caveolin-1. In the control sample exposed to biot.-rmMCP-1 at 37°C, caveolin-1 staining can be seen concentrated around the periphery of the microvascular segment ( arrows ), with chemokine apparently present diffusely in the cytoplasm. Areas of yellow fluorescence ( asterisks ) indicate presumed sites of biot.-rmMCP-1/caveolin-1 colocalization. In the filipin III-treated sample exposed to chemokine at 37°C, no sites of colocalization are detected readily, and biot.-rmMCP-1 appears to be confined to the membrane surface ( arrowheads ), with caveolin-1 expression heightened in some cytoplasmic locales ( arrows ). Microvessels exposed to chemokine at 4°C (±) filipin III pretreatment also fail to show any areas of biot.-rmMCP-1/caveolin-1 colocalization. These samples also do not demonstrate as strong a cytoplasmic distribution of labeled chemokine as that observed in the control sample at 37°C but seemingly manifest a more peripheral chemokine staining ( arrowheads ), possibly restricted to the membrane surface. As with the samples exposed to chemokine at 37°C, caveolin-1 distribution appears to be concentrated along the periphery of the microvessel in the control ( arrowheads ) but is dispersed more cytoplasmically in the filipin III-treated sample. Arrows , Caveolin-1; arrowheads , biot.-rmMCP-1; asterisks , biot.-rmMCP-1/caveolin-1 colocalization. Scale bar, 10 μm.

    Techniques Used: Staining, Labeling, Fluorescence, Expressing

    40) Product Images from "Dynamin2, Clathrin, and Lipid Rafts Mediate Endocytosis of the Apical Na/K/2Cl Cotransporter NKCC2 in Thick Ascending Limbs *"

    Article Title: Dynamin2, Clathrin, and Lipid Rafts Mediate Endocytosis of the Apical Na/K/2Cl Cotransporter NKCC2 in Thick Ascending Limbs *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.386425

    Effect of clathrin inhibition and lipid raft disruption on NKCC2 retrieval in THALs. A , cumulative data showing the effect of simultaneous treatment with the clathrin inhibitor chlorpromazine (20 μ m ) and lipid-raft disrupting agent filipin III
    Figure Legend Snippet: Effect of clathrin inhibition and lipid raft disruption on NKCC2 retrieval in THALs. A , cumulative data showing the effect of simultaneous treatment with the clathrin inhibitor chlorpromazine (20 μ m ) and lipid-raft disrupting agent filipin III

    Techniques Used: Inhibition

    Effect of lipid raft disruption on NKCC2 retrieval in THALs. A , cumulative data showing the effect of filipin III (2.5 μ m ) on constitutive NKCC2 endocytosis in THALs. Control or THALs treated with filipin III (2.5 μ m ) were biotinylated
    Figure Legend Snippet: Effect of lipid raft disruption on NKCC2 retrieval in THALs. A , cumulative data showing the effect of filipin III (2.5 μ m ) on constitutive NKCC2 endocytosis in THALs. Control or THALs treated with filipin III (2.5 μ m ) were biotinylated

    Techniques Used:

    41) Product Images from "Tumor cell-released autophagosomes (TRAP) enhance apoptosis and immunosuppressive functions of neutrophils"

    Article Title: Tumor cell-released autophagosomes (TRAP) enhance apoptosis and immunosuppressive functions of neutrophils

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1438108

    Neutrophil apoptosis requires TRAP internalization by macropinocytosis. (A) Neutrophils were co-incubated with CFSE-labeled TRAPs (3 μg/ml) for 6 h and stained with Annexin V-Alexa Fluor 647. The percentage of CFSE + neutrophils and Annexin V + neutrophils were analyzed by flow cytometry. (B) Neutrophils were pre-treated with CD (10 μg/ml), CPZ (4 μg/ml) or filipin III (12 μg/ml) for 30 min, and then co-cultured with TRAPs (3 μg/ml) for 3 h. The phagocytizing of TRAPs by neutrophils was detected by flow cytometry. (C) Neutrophils were pre-treated with CD (10 μg/ml), CPZ (4 μg/ml) or filipin III (12 μg/ml) for 30 min, co-incubated with or without TRAPs (10 μg/ml) for 6 h, and then the apoptosis of neutrophils was assessed by flow cytometry. (D) Neutrophils were pre-treated with EIPA (20 μM) for 30 min, and then co-cultured with TRAPs (3 μg/ml) for 3 h. The phagocytizing of TRAPs by neutrophils was analyzed by flow cytometry. (E) Neutrophils were pre-treated with EIPA (20 μM) for 30 min, co-incubated with or without TRAPs (10 μg/ml) for 6 h, and then the apoptosis of neutrophils was assessed by flow cytometry. Results are representative of at least three independent experiments. ** p
    Figure Legend Snippet: Neutrophil apoptosis requires TRAP internalization by macropinocytosis. (A) Neutrophils were co-incubated with CFSE-labeled TRAPs (3 μg/ml) for 6 h and stained with Annexin V-Alexa Fluor 647. The percentage of CFSE + neutrophils and Annexin V + neutrophils were analyzed by flow cytometry. (B) Neutrophils were pre-treated with CD (10 μg/ml), CPZ (4 μg/ml) or filipin III (12 μg/ml) for 30 min, and then co-cultured with TRAPs (3 μg/ml) for 3 h. The phagocytizing of TRAPs by neutrophils was detected by flow cytometry. (C) Neutrophils were pre-treated with CD (10 μg/ml), CPZ (4 μg/ml) or filipin III (12 μg/ml) for 30 min, co-incubated with or without TRAPs (10 μg/ml) for 6 h, and then the apoptosis of neutrophils was assessed by flow cytometry. (D) Neutrophils were pre-treated with EIPA (20 μM) for 30 min, and then co-cultured with TRAPs (3 μg/ml) for 3 h. The phagocytizing of TRAPs by neutrophils was analyzed by flow cytometry. (E) Neutrophils were pre-treated with EIPA (20 μM) for 30 min, co-incubated with or without TRAPs (10 μg/ml) for 6 h, and then the apoptosis of neutrophils was assessed by flow cytometry. Results are representative of at least three independent experiments. ** p

    Techniques Used: Incubation, Labeling, Staining, Flow Cytometry, Cytometry, Cell Culture

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

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

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M066522

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

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

    43) Product Images from "The Prion Protein N1 and N2 Cleavage Fragments Bind to Phosphatidylserine and Phosphatidic Acid; Relevance to Stress-Protection Responses"

    Article Title: The Prion Protein N1 and N2 Cleavage Fragments Bind to Phosphatidylserine and Phosphatidic Acid; Relevance to Stress-Protection Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134680

    Serum deprivation causes changes to PS and PA in CF10 cells. A. Laurdan GP changes following serum deprivation for 30 minutes as compared with benzyl alcohol (BA) and filipin III controls. n = 3. B. Live cell imaging of NBD-PS labelled CF10 cells. Image intensity is thresholds have been selected to view detail in the staining pattern and do not represent a comparison of fluorescence intensity. Scale bars = 20 μm. C. Fluorescence emission spectra of NBD-PS labelled cells following transfer into serum-free medium, scans were taken immediately after media replacement. D. Anisotropy of NBD-PS in CF10 cells with and without serum present. n = 3. E. Counts from magnetic separation of cells that have lost membrane asymmetry allowing them to bind PS at 5 and 15 minutes post serum withdrawal. n = 4. F. ROS production detected by DCF fluorescence when cells are serum-starved and with exposure to butan-1-ol to inhibit PLD activity. n = 4. G. Measurement of cellular phosphatidic acid concentration 30 minutes after commencing serum deprivation. n = 3. H. Measurement of phospholipase-D activity following 15 minutes serum starvation. n = 3. For all panels, *p
    Figure Legend Snippet: Serum deprivation causes changes to PS and PA in CF10 cells. A. Laurdan GP changes following serum deprivation for 30 minutes as compared with benzyl alcohol (BA) and filipin III controls. n = 3. B. Live cell imaging of NBD-PS labelled CF10 cells. Image intensity is thresholds have been selected to view detail in the staining pattern and do not represent a comparison of fluorescence intensity. Scale bars = 20 μm. C. Fluorescence emission spectra of NBD-PS labelled cells following transfer into serum-free medium, scans were taken immediately after media replacement. D. Anisotropy of NBD-PS in CF10 cells with and without serum present. n = 3. E. Counts from magnetic separation of cells that have lost membrane asymmetry allowing them to bind PS at 5 and 15 minutes post serum withdrawal. n = 4. F. ROS production detected by DCF fluorescence when cells are serum-starved and with exposure to butan-1-ol to inhibit PLD activity. n = 4. G. Measurement of cellular phosphatidic acid concentration 30 minutes after commencing serum deprivation. n = 3. H. Measurement of phospholipase-D activity following 15 minutes serum starvation. n = 3. For all panels, *p

    Techniques Used: Live Cell Imaging, Staining, Fluorescence, Activity Assay, Concentration Assay

    44) Product Images from "Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells"

    Article Title: Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells

    Journal: Journal of Breast Cancer

    doi: 10.4048/jbc.2016.19.4.372

    Effect of cholesterol depleting agents on cytotoxicity of triple negative breast cancer cells. MDA-MB 231 and 468 cells were treated with methyl-β-cyclodextrin (MβCD), nystatin and filipin III at different concentrations (0.1–0.5 mM) for 1, 24, and 48 hours and cell proliferation in terms of cytotoxicity was measured using lactate dehydrogenase assay. Cytotoxic effect of MβCD (A, B), nystatin (C, D) and filipin III (E, F) in MDA-MB 231 and MDA-MB 468 cells, respectively. The cytotoxicity was expressed as percent control. The results represent the mean±SD of three independent experiments.
    Figure Legend Snippet: Effect of cholesterol depleting agents on cytotoxicity of triple negative breast cancer cells. MDA-MB 231 and 468 cells were treated with methyl-β-cyclodextrin (MβCD), nystatin and filipin III at different concentrations (0.1–0.5 mM) for 1, 24, and 48 hours and cell proliferation in terms of cytotoxicity was measured using lactate dehydrogenase assay. Cytotoxic effect of MβCD (A, B), nystatin (C, D) and filipin III (E, F) in MDA-MB 231 and MDA-MB 468 cells, respectively. The cytotoxicity was expressed as percent control. The results represent the mean±SD of three independent experiments.

    Techniques Used: Multiple Displacement Amplification, Lactate Dehydrogenase Assay

    Effect of cholesterol depleting agents on membrane cholesterol in triple negative breast cancer cells. MDA-MB 231 and 468 cells were treated with methyl-β-cyclodextrin (MβCD), nystatin and filipin III at different concentrations (0.1–0.5 mM) for 1, 24, and 48 hours and reduction in cellular cholesterol levels were measured. Reduction of cellular cholesterol with MβCD (A, B), nystatin (C, D) and filipin III (E, F) in MDA-MB 231 and MDA-MB 468 cells, respectively. The percent reduction in the cholesterol upon the treatments was calculated with respect to total cholesterol in the untreated cells, which was taken as 100%. The results represent the mean±SD of three independent experiments.
    Figure Legend Snippet: Effect of cholesterol depleting agents on membrane cholesterol in triple negative breast cancer cells. MDA-MB 231 and 468 cells were treated with methyl-β-cyclodextrin (MβCD), nystatin and filipin III at different concentrations (0.1–0.5 mM) for 1, 24, and 48 hours and reduction in cellular cholesterol levels were measured. Reduction of cellular cholesterol with MβCD (A, B), nystatin (C, D) and filipin III (E, F) in MDA-MB 231 and MDA-MB 468 cells, respectively. The percent reduction in the cholesterol upon the treatments was calculated with respect to total cholesterol in the untreated cells, which was taken as 100%. The results represent the mean±SD of three independent experiments.

    Techniques Used: Multiple Displacement Amplification

    45) 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

    46) Product Images from "Upregulation of MUC5AC by VEGF in human primary bronchial epithelial cells: implications for asthma"

    Article Title: Upregulation of MUC5AC by VEGF in human primary bronchial epithelial cells: implications for asthma

    Journal: Respiratory Research

    doi: 10.1186/s12931-019-1245-1

    VEGFR2 association with caveolin-1 is markedly decreased by treatment with VEGF, cyclodextrin, or filipin and is partially rescued by treatment with cholesterol repletion. VEGFR2 was immunopricipitated from PBECs lysates, and its association with caveolin-1 was assessed by Western blot. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions (A). PBECs were treated with VEGF (50 ng/ml) in the presence or absence of pretreatment with the caveolar-disrupting agent cyclodextrin (CD; 5 mM, 30 min) or filipin (2.5 g/ml, 30 min). Reversal of drug effects was sought with simultaneous cholesterol repletion (Chol; 15 g/ml) given at the time of cyclodextrin administration. Caveolin-1was immunopricipitated from PBECs lysates, and its association with VEGFR2 was assessed by Western blot. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions (B). All data are representative of three independent experiments
    Figure Legend Snippet: VEGFR2 association with caveolin-1 is markedly decreased by treatment with VEGF, cyclodextrin, or filipin and is partially rescued by treatment with cholesterol repletion. VEGFR2 was immunopricipitated from PBECs lysates, and its association with caveolin-1 was assessed by Western blot. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions (A). PBECs were treated with VEGF (50 ng/ml) in the presence or absence of pretreatment with the caveolar-disrupting agent cyclodextrin (CD; 5 mM, 30 min) or filipin (2.5 g/ml, 30 min). Reversal of drug effects was sought with simultaneous cholesterol repletion (Chol; 15 g/ml) given at the time of cyclodextrin administration. Caveolin-1was immunopricipitated from PBECs lysates, and its association with VEGFR2 was assessed by Western blot. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions (B). All data are representative of three independent experiments

    Techniques Used: Western Blot, Immunoprecipitation

    Caveolar disruption enhanced the VEGF-induced RhoA activation and MUC5AC up-regulation. PBECs were treated with VEGF (50 ng/ml) in the presence or absence of pretreatment with the caveolar-disrupting agent cyclodextrin (CD; 5 mM, 30 min) or filipin (2.5 g/ml, 30 min). Reversal of drug effects was sought with simultaneous cholesterol repletion (Chol; 15 g/ml) given at the time of cyclodextrin administration. RhoA activity was assessed by pull-down assay of GTP-bound RhoA (24 kDa) as described in Method (A). MUC5AC protein levels were assessed by Western blot (B). All data are representative of three independent experiments. The blots were quantified by densitometry. Values represent the means ± SEM. * P
    Figure Legend Snippet: Caveolar disruption enhanced the VEGF-induced RhoA activation and MUC5AC up-regulation. PBECs were treated with VEGF (50 ng/ml) in the presence or absence of pretreatment with the caveolar-disrupting agent cyclodextrin (CD; 5 mM, 30 min) or filipin (2.5 g/ml, 30 min). Reversal of drug effects was sought with simultaneous cholesterol repletion (Chol; 15 g/ml) given at the time of cyclodextrin administration. RhoA activity was assessed by pull-down assay of GTP-bound RhoA (24 kDa) as described in Method (A). MUC5AC protein levels were assessed by Western blot (B). All data are representative of three independent experiments. The blots were quantified by densitometry. Values represent the means ± SEM. * P

    Techniques Used: Activation Assay, Activity Assay, Pull Down Assay, Western Blot

    47) Product Images from "Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KII? at the trans-Golgi network"

    Article Title: Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KII? at the trans-Golgi network

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M005751

    Distribution of sterol and endogenous PI4KIIα in A431 cells. Fixed cells were costained with filipin III (green channel) and an anti-PI4KIIα monoclonal antibody (red channel) and imaged by confocal microscopy. Arrows indicate membranes
    Figure Legend Snippet: Distribution of sterol and endogenous PI4KIIα in A431 cells. Fixed cells were costained with filipin III (green channel) and an anti-PI4KIIα monoclonal antibody (red channel) and imaged by confocal microscopy. Arrows indicate membranes

    Techniques Used: Confocal Microscopy

    48) Product Images from "Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway"

    Article Title: Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-019-0444-8

    Effects of several concentrations of inhibitors, filipin III ( a ), β-cyclodextrin ( b ), and chlorpromazine ( c ), on internalization of siRNA with/without INT and mTat/PEI/INT in HSC-3 cells. β - actin mRNA was measured by QRT-PCR and then % remaining β - actin mRNA expression was calculated based on control as 100%. * p
    Figure Legend Snippet: Effects of several concentrations of inhibitors, filipin III ( a ), β-cyclodextrin ( b ), and chlorpromazine ( c ), on internalization of siRNA with/without INT and mTat/PEI/INT in HSC-3 cells. β - actin mRNA was measured by QRT-PCR and then % remaining β - actin mRNA expression was calculated based on control as 100%. * p

    Techniques Used: Quantitative RT-PCR, Expressing

    49) Product Images from "The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells"

    Article Title: The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192250

    Localization of Slp3p. (A) Exponential-phase samples of the untagged wt SLP3 control strain and (B) SLP3-YFP expressing strain were treated with 160 μM FM 4–64. The FM 4–64 incubation period was set to allow visualization of the vacuole. Cells were viewed under bright-field (BF) and fluorescent microscopy. The right panels show an overlay of the YFP and FM 4–64 fluorescence (YFP/FM 4–64). (C) Overlay photo shown in (B) was recolored with ImageJ to assess Slp3p-Yfp vacuolar membrane localization. The yellow fluorescence color was reassigned to green. (D) Overnight-cultured SLP3-YFP cells were treated with 200 μg/mL Filipin and viewed using fluorescent microscopy. Image depicts a single cell, and the right panel shows an overlay of the YFP and Filipin fluorescence (YFP/Filipin). The dashed white box highlights the cell depicted in the inset image. For each assay, three biological replicates were analyzed. Experiments were repeated at least three times, and data presented represents one representative experiment. Approximately 1.0 x 10 4 cells of each strain were selected for viewing (A-D). Scale bars represent 10 μm.
    Figure Legend Snippet: Localization of Slp3p. (A) Exponential-phase samples of the untagged wt SLP3 control strain and (B) SLP3-YFP expressing strain were treated with 160 μM FM 4–64. The FM 4–64 incubation period was set to allow visualization of the vacuole. Cells were viewed under bright-field (BF) and fluorescent microscopy. The right panels show an overlay of the YFP and FM 4–64 fluorescence (YFP/FM 4–64). (C) Overlay photo shown in (B) was recolored with ImageJ to assess Slp3p-Yfp vacuolar membrane localization. The yellow fluorescence color was reassigned to green. (D) Overnight-cultured SLP3-YFP cells were treated with 200 μg/mL Filipin and viewed using fluorescent microscopy. Image depicts a single cell, and the right panel shows an overlay of the YFP and Filipin fluorescence (YFP/Filipin). The dashed white box highlights the cell depicted in the inset image. For each assay, three biological replicates were analyzed. Experiments were repeated at least three times, and data presented represents one representative experiment. Approximately 1.0 x 10 4 cells of each strain were selected for viewing (A-D). Scale bars represent 10 μm.

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

    50) Product Images from "Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells"

    Article Title: Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells

    Journal: BioMed Research International

    doi: 10.1155/2014/687835

    Adhesion and internalization percentages with (a) LLC-MK2 cells and (b) murine macrophages after filipin treatment (1, 3, and 6 nM) for 30 min before the addition of the parasites (50 : 1) for 10 minutes. At 6 nM filipin, the adhesion of the parasites to the LLC-MK2 cells was slightly decreased, and internalization was significantly inhibited. In macrophages, internalization was inhibited by 85%. The data shown are the means ± SE of duplicated points from three independent experiments. * P
    Figure Legend Snippet: Adhesion and internalization percentages with (a) LLC-MK2 cells and (b) murine macrophages after filipin treatment (1, 3, and 6 nM) for 30 min before the addition of the parasites (50 : 1) for 10 minutes. At 6 nM filipin, the adhesion of the parasites to the LLC-MK2 cells was slightly decreased, and internalization was significantly inhibited. In macrophages, internalization was inhibited by 85%. The data shown are the means ± SE of duplicated points from three independent experiments. * P

    Techniques Used:

    51) Product Images from "Endocytic Crosstalk: Cavins, Caveolins, and Caveolae Regulate Clathrin-Independent Endocytosis"

    Article Title: Endocytic Crosstalk: Cavins, Caveolins, and Caveolae Regulate Clathrin-Independent Endocytosis

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001832

    CAV1 alters general membrane characteristics. (A) WT and CAV1−/− MEFs were transiently transfected with GP1-YFP; CAV1−/− MEFs were also co-transfected with GPI-YFP plus CAV1-mCherry or CAV1G83S-mCherry. FRAP analysis for GPI-YFP diffusion was performed, and FRAP curves and diffusion coefficients (D) calculated from FRAP measurements in at least 30 cells per condition are shown. (B) CAV1−/− MEFs were transiently transfected with PA-CD44 and co-transfected with PA-CD44 and CAV1 respectively. Fluorescence decay curves and diffusion coefficients (D) for PA-CD44, calculated from photo-activation measurements in at least 20 cells per condition, are shown. (C) CAV1−/− MEFs were either treated with 0.05% Tween20 at 37°C prior to performing internalization assay with anti-CD44 mAb and Tfn-647 or incubated with endocytic markers at 41°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 40–50 cells from each treatment were quantified for normalized fluorescence intensity of endocytic markers. (D,E) CAV1−/− MEFs transiently expressing CAV1-YFP and Cavin-1-GFP respectively, were either treated with Tween20 or incubated with endocytic markers at 41°C. Uptake and quantification of internalized markers was performed as mentioned in (C). (F) In CAV1−/− MEFs transiently expressing CAV1-YFP, internalization assay were performed with Dex-488 for 5 min either at 41°C or at 37°C. 40–50 cells from each condition were quantified for fluorescence intensity of the internalized Dex-488. (G) Representative images for filipin labeling performed for CAV1-YFP-expressing treated and untreated CAV1−/− MEFs and a bar graph representing quantification for normalized fluorescence intensity of filipin are shown. In (A) and (B) data represent average mean ± SEM of pooled data from three independent experiments. Diffusion coefficients were obtained by non-linear regression of recovery and fluorescence decay curves, as described in the Materials and Methods section. In (C–F) data represent mean ± SEM of three independent experiments. *p
    Figure Legend Snippet: CAV1 alters general membrane characteristics. (A) WT and CAV1−/− MEFs were transiently transfected with GP1-YFP; CAV1−/− MEFs were also co-transfected with GPI-YFP plus CAV1-mCherry or CAV1G83S-mCherry. FRAP analysis for GPI-YFP diffusion was performed, and FRAP curves and diffusion coefficients (D) calculated from FRAP measurements in at least 30 cells per condition are shown. (B) CAV1−/− MEFs were transiently transfected with PA-CD44 and co-transfected with PA-CD44 and CAV1 respectively. Fluorescence decay curves and diffusion coefficients (D) for PA-CD44, calculated from photo-activation measurements in at least 20 cells per condition, are shown. (C) CAV1−/− MEFs were either treated with 0.05% Tween20 at 37°C prior to performing internalization assay with anti-CD44 mAb and Tfn-647 or incubated with endocytic markers at 41°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 40–50 cells from each treatment were quantified for normalized fluorescence intensity of endocytic markers. (D,E) CAV1−/− MEFs transiently expressing CAV1-YFP and Cavin-1-GFP respectively, were either treated with Tween20 or incubated with endocytic markers at 41°C. Uptake and quantification of internalized markers was performed as mentioned in (C). (F) In CAV1−/− MEFs transiently expressing CAV1-YFP, internalization assay were performed with Dex-488 for 5 min either at 41°C or at 37°C. 40–50 cells from each condition were quantified for fluorescence intensity of the internalized Dex-488. (G) Representative images for filipin labeling performed for CAV1-YFP-expressing treated and untreated CAV1−/− MEFs and a bar graph representing quantification for normalized fluorescence intensity of filipin are shown. In (A) and (B) data represent average mean ± SEM of pooled data from three independent experiments. Diffusion coefficients were obtained by non-linear regression of recovery and fluorescence decay curves, as described in the Materials and Methods section. In (C–F) data represent mean ± SEM of three independent experiments. *p

    Techniques Used: Transfection, Diffusion-based Assay, Fluorescence, Activation Assay, Incubation, Labeling, Expressing

    Cavin-1 regulates cholesterol distribution and Cdc42 activity. (A) Filipin labeling was performed for Cavin-1-GFP-expressing CAV1−/− MEFs, and for quantification average fluorescence intensity measured from 40–50 transfected cells was normalized to untransfected cells. (B) Whole cell lysates from CAV1−/− MEFs expressing CAV1-YFP and Cavin-1-GFP respectively were immunoblotted for Cdc42 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The bar graph represents quantitation of protein levels, calculated by measuring band intensities by densitometry. (C) Pearson coefficient analysis determined at the plasma membrane for CAV1/Cavin-1 (positive control), Cavin-1/Cdc42, and randomized Cavin-1/Cdc42. (D) CAV1−/− MEFs were co-transfected with CRIB-YPet and Cavin-1-mCherry and quantitative colocalization analysis was performed at the PM ruffles. For 30 cells, line scans (10 pixels' length) were performed at random positions on PM ruffles and cytosol. The resultant fluorescent intensities profiles were plotted against each other and subjected to linear regression analysis. (E) CAV1−/− MEFs were transiently transfected with Cdc42-CyPet alone, with Cdc42-CyPet and CRIB-YPet simultaneously, or with Cdc42-CyPet, CRIB-YPet, and Cavin-1-Flag. Lifetime of CyPet was analyzed by FLIM-FRET and a representative graph showing lifetime of CyPet in 25 cells, mean ± SEM, is shown. (F) In Cavin-1−/− and CAV1−/− MEFs internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 and 10 min at 37°C. Endogenous CAV1 and Cavin-1 was labeled with respective primary antibodies followed by AF-488 secondary antibody labeling and for internalized anti-CD44 mAb labeling AF-555 secondary antibody was used. In (A,B,D) data represent mean ± SEM of three independent experiments. In (C) data represent average mean ± SEM of pooled data from three independent experiments; n = 40 cells. **p
    Figure Legend Snippet: Cavin-1 regulates cholesterol distribution and Cdc42 activity. (A) Filipin labeling was performed for Cavin-1-GFP-expressing CAV1−/− MEFs, and for quantification average fluorescence intensity measured from 40–50 transfected cells was normalized to untransfected cells. (B) Whole cell lysates from CAV1−/− MEFs expressing CAV1-YFP and Cavin-1-GFP respectively were immunoblotted for Cdc42 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The bar graph represents quantitation of protein levels, calculated by measuring band intensities by densitometry. (C) Pearson coefficient analysis determined at the plasma membrane for CAV1/Cavin-1 (positive control), Cavin-1/Cdc42, and randomized Cavin-1/Cdc42. (D) CAV1−/− MEFs were co-transfected with CRIB-YPet and Cavin-1-mCherry and quantitative colocalization analysis was performed at the PM ruffles. For 30 cells, line scans (10 pixels' length) were performed at random positions on PM ruffles and cytosol. The resultant fluorescent intensities profiles were plotted against each other and subjected to linear regression analysis. (E) CAV1−/− MEFs were transiently transfected with Cdc42-CyPet alone, with Cdc42-CyPet and CRIB-YPet simultaneously, or with Cdc42-CyPet, CRIB-YPet, and Cavin-1-Flag. Lifetime of CyPet was analyzed by FLIM-FRET and a representative graph showing lifetime of CyPet in 25 cells, mean ± SEM, is shown. (F) In Cavin-1−/− and CAV1−/− MEFs internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 and 10 min at 37°C. Endogenous CAV1 and Cavin-1 was labeled with respective primary antibodies followed by AF-488 secondary antibody labeling and for internalized anti-CD44 mAb labeling AF-555 secondary antibody was used. In (A,B,D) data represent mean ± SEM of three independent experiments. In (C) data represent average mean ± SEM of pooled data from three independent experiments; n = 40 cells. **p

    Techniques Used: Activity Assay, Labeling, Expressing, Fluorescence, Transfection, Quantitation Assay, Positive Control, Antibody Labeling

    52) Product Images from "A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux"

    Article Title: A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux

    Journal: ACS Nano

    doi: 10.1021/acsnano.7b04743

    Measurement of endolysosomal lipid accumulation and reversal in NPC1 patient-derived fibroblasts. (a) Mean reporter emission from wild-type fibroblasts, patient-derived NPC1 fibroblasts, and NPC1 fibroblasts treated with hydroxypropyl-β-cyclodextrin (HPβCD) for 24 h. Statistical significance was determined with a one-way ANOVA with Sidak’s multiple comparison test. (b) Histograms of the nanotube emission wavelength from single endolysosomal organelles of wild-type fibroblasts, NPC1 patient fibroblasts, and NPC1 fibroblasts treated with HPβCD for 24 h. (c) Mean filipin intensity from WT fibroblasts, NPC1 cells, and NPC1 cells treated with HPβCD for 24 h, at 48 h after nanotube addition. Statistical significance was determined with a one-way ANOVA with Tukey’s multiple comparison test. All error bars are SEM from three technical replicate experiments. * = p
    Figure Legend Snippet: Measurement of endolysosomal lipid accumulation and reversal in NPC1 patient-derived fibroblasts. (a) Mean reporter emission from wild-type fibroblasts, patient-derived NPC1 fibroblasts, and NPC1 fibroblasts treated with hydroxypropyl-β-cyclodextrin (HPβCD) for 24 h. Statistical significance was determined with a one-way ANOVA with Sidak’s multiple comparison test. (b) Histograms of the nanotube emission wavelength from single endolysosomal organelles of wild-type fibroblasts, NPC1 patient fibroblasts, and NPC1 fibroblasts treated with HPβCD for 24 h. (c) Mean filipin intensity from WT fibroblasts, NPC1 cells, and NPC1 cells treated with HPβCD for 24 h, at 48 h after nanotube addition. Statistical significance was determined with a one-way ANOVA with Tukey’s multiple comparison test. All error bars are SEM from three technical replicate experiments. * = p

    Techniques Used: Derivative Assay

    53) Product Images from "Serelaxin-mediated signal transduction in human vascular cells: bell-shaped concentration–response curves reflect differential coupling to G proteins"

    Article Title: Serelaxin-mediated signal transduction in human vascular cells: bell-shaped concentration–response curves reflect differential coupling to G proteins

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12964

    The role of G-protein coupling in serelaxin-mediated cGMP accumulation in human primary umbilical vascular cells. Pretreatment of HUASMC ( n = 6) in (A) and of HUVSMC ( n = 6) in (B) with the selective Gα s inhibitor NF449 (10 μM, 30 min) caused a rightward shift and reduced the E-max of the cGMP CRC to serelaxin without modifying the shape of the curve. Pretreatment of HUASMC ( n = 7) in (C) and of HUVSMC ( n = 6) in (D) with the selective Gα i/o inhibitor NF023 (10 μM, 30 min) reduced the E-max of the cGMP CRC to serelaxin and changed the shape of the curve observed with HUVSMC (D) from bell-shaped to sigmoidal. In both (E) HUASMC ( n = 6) and (F) HUVSMC ( n = 6), pretreatment with both NF449 and NF023 completely abolished serelaxin-mediated cGMP responses, showing that the responses result entirely from RXFP1 receptors interaction with G proteins. In (G) HUASMC ( n = 6) and in (H) HUVSMC ( n = 6), pretreatment with filipin III (1 μg·mL −1 , 1 h), which disrupts lipid rafts, mimicked the effect of the Gα i/o inhibitor NF023 – reducing E-max and converting CRCs from bell-shaped to sigmoidal in HUVSMC.
    Figure Legend Snippet: The role of G-protein coupling in serelaxin-mediated cGMP accumulation in human primary umbilical vascular cells. Pretreatment of HUASMC ( n = 6) in (A) and of HUVSMC ( n = 6) in (B) with the selective Gα s inhibitor NF449 (10 μM, 30 min) caused a rightward shift and reduced the E-max of the cGMP CRC to serelaxin without modifying the shape of the curve. Pretreatment of HUASMC ( n = 7) in (C) and of HUVSMC ( n = 6) in (D) with the selective Gα i/o inhibitor NF023 (10 μM, 30 min) reduced the E-max of the cGMP CRC to serelaxin and changed the shape of the curve observed with HUVSMC (D) from bell-shaped to sigmoidal. In both (E) HUASMC ( n = 6) and (F) HUVSMC ( n = 6), pretreatment with both NF449 and NF023 completely abolished serelaxin-mediated cGMP responses, showing that the responses result entirely from RXFP1 receptors interaction with G proteins. In (G) HUASMC ( n = 6) and in (H) HUVSMC ( n = 6), pretreatment with filipin III (1 μg·mL −1 , 1 h), which disrupts lipid rafts, mimicked the effect of the Gα i/o inhibitor NF023 – reducing E-max and converting CRCs from bell-shaped to sigmoidal in HUVSMC.

    Techniques Used:

    The role of G-protein coupling in serelaxin-mediated cAMP accumulation in human primary umbilical vascular cells. Pretreatment of HUASMC ( n = 6) in (A) and of HUVSMC ( n = 6) in (B) with the selective Gα s inhibitor NF449 (10 μM, 30 min) caused a rightward shift and reduced the E-max of the cAMP CRC to serelaxin without modifying the shape of the curve. Pretreatment of HUASMC ( n = 7) in (C) and of HUVSMC ( n = 6) in (D) with the selective Gα i/o inhibitor NF023 (10 μM, 30 min) reduced the E-max of the cAMP CRC to serelaxin and changed the shape of the curve observed with HUVSMC (D) from bell-shaped to sigmoidal. In both (E) HUASMC ( n = 6) and (F) HUVSMC ( n = 6), pretreatment with both NF449 and NF023 completely abolished serelaxin-mediated cAMP responses, showing that the responses result entirely from RXFP1 receptors interaction with G proteins. In (G) HUASMC ( n = 6) and in (H) HUVSMC ( n = 6), pretreatment with filipin III (1 μg·mL −1 , 1 h), which disrupts lipid rafts, mimicked the effect of the Gα i/o inhibitor NF023 – reducing E-max and converting CRCs from bell-shaped to sigmoidal in HUVSMC.
    Figure Legend Snippet: The role of G-protein coupling in serelaxin-mediated cAMP accumulation in human primary umbilical vascular cells. Pretreatment of HUASMC ( n = 6) in (A) and of HUVSMC ( n = 6) in (B) with the selective Gα s inhibitor NF449 (10 μM, 30 min) caused a rightward shift and reduced the E-max of the cAMP CRC to serelaxin without modifying the shape of the curve. Pretreatment of HUASMC ( n = 7) in (C) and of HUVSMC ( n = 6) in (D) with the selective Gα i/o inhibitor NF023 (10 μM, 30 min) reduced the E-max of the cAMP CRC to serelaxin and changed the shape of the curve observed with HUVSMC (D) from bell-shaped to sigmoidal. In both (E) HUASMC ( n = 6) and (F) HUVSMC ( n = 6), pretreatment with both NF449 and NF023 completely abolished serelaxin-mediated cAMP responses, showing that the responses result entirely from RXFP1 receptors interaction with G proteins. In (G) HUASMC ( n = 6) and in (H) HUVSMC ( n = 6), pretreatment with filipin III (1 μg·mL −1 , 1 h), which disrupts lipid rafts, mimicked the effect of the Gα i/o inhibitor NF023 – reducing E-max and converting CRCs from bell-shaped to sigmoidal in HUVSMC.

    Techniques Used:

    54) Product Images from "Cyclodextrins: Assessing the Impact of Cavity Size, Occupancy, and Substitutions on Cytotoxicity and Cholesterol Homeostasis"

    Article Title: Cyclodextrins: Assessing the Impact of Cavity Size, Occupancy, and Substitutions on Cytotoxicity and Cholesterol Homeostasis

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23051228

    Effect of CD derivatives on the intracellular levels of free form of cholesterol. Primary fibroblast cells from a healthy donor or Niemann–Pick disease type C (NPC) patient were incubated with CD derivatives (1 mM) for 72 h, and the levels of free cholesterol were determined by staining with Filipin III. Data shown are representative of three independent experiments. Wild type, primary fibroblast cells from a healthy donor; NPC1 mutant, primary fibroblast cells from an NPC patient. The size of a scale bar is 200 μm.
    Figure Legend Snippet: Effect of CD derivatives on the intracellular levels of free form of cholesterol. Primary fibroblast cells from a healthy donor or Niemann–Pick disease type C (NPC) patient were incubated with CD derivatives (1 mM) for 72 h, and the levels of free cholesterol were determined by staining with Filipin III. Data shown are representative of three independent experiments. Wild type, primary fibroblast cells from a healthy donor; NPC1 mutant, primary fibroblast cells from an NPC patient. The size of a scale bar is 200 μm.

    Techniques Used: Incubation, Staining, Mutagenesis

    55) Product Images from "Caveolin-1 Controls Vesicular TLR2 Expression, p38 Signaling and T Cell Suppression in BCG Infected Murine Monocytic Myeloid-Derived Suppressor Cells"

    Article Title: Caveolin-1 Controls Vesicular TLR2 Expression, p38 Signaling and T Cell Suppression in BCG Infected Murine Monocytic Myeloid-Derived Suppressor Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02826

    Pharmacological inhibition or genetic deficiency of Cav-1 do not impair BCG uptake by G-MDSC and M-MDSC. (A) MDSCs were left untreated or pretreated or not with Cytochalasin D for 1 h and then incubated with BCG-GFP at MOI 2 for 6 h. Cells were then analyzed by flow cytometry for % BCG uptake by GFP detection (gated by dotted line) in G-MDSC and M-MDSC subsets by gates shown in Figure 1A . Dotted lines indicate gating for positive detection of uptake by GFP fluorescence. (B) As in A but several pooled experiments are shown and MDSCs were incubated with cytochalasin-D, filipin III, simvastatin or β-cyclodextrine for 1 h and then stimulated with BCG-GFP at MOI of 2, 5, or 10 for 6 h. (C,D) MDSCs of WT and Cav1 −/− mice were incubated with BCG-GFP at MOI 2 for 6 h. Cells were then analyzed by flow cytometry for BCG uptake by GFP detection in G-MDSC and M-MDSC subsets by gating as shown in Figure 1A . Pooled and normalized data of several experiments as performed for (A) . (E) MDSCs were stimulated with BCG-GFP at 1 MOI for 16 h. Cytospins were then stained for Cav-1 and DAPI and analyzed by confocal microcopy. G-MDSC and M-MDSCs were defined on the basis of their nuclear shape. Scale bars upper row 7 μm, all other rows 10 μm. (F) Pearson's correlation coefficients for colocalization (“overlap”) of BCG and Cav-1 from n = 5 independent experiments like shown in (E) . Data are from **** P
    Figure Legend Snippet: Pharmacological inhibition or genetic deficiency of Cav-1 do not impair BCG uptake by G-MDSC and M-MDSC. (A) MDSCs were left untreated or pretreated or not with Cytochalasin D for 1 h and then incubated with BCG-GFP at MOI 2 for 6 h. Cells were then analyzed by flow cytometry for % BCG uptake by GFP detection (gated by dotted line) in G-MDSC and M-MDSC subsets by gates shown in Figure 1A . Dotted lines indicate gating for positive detection of uptake by GFP fluorescence. (B) As in A but several pooled experiments are shown and MDSCs were incubated with cytochalasin-D, filipin III, simvastatin or β-cyclodextrine for 1 h and then stimulated with BCG-GFP at MOI of 2, 5, or 10 for 6 h. (C,D) MDSCs of WT and Cav1 −/− mice were incubated with BCG-GFP at MOI 2 for 6 h. Cells were then analyzed by flow cytometry for BCG uptake by GFP detection in G-MDSC and M-MDSC subsets by gating as shown in Figure 1A . Pooled and normalized data of several experiments as performed for (A) . (E) MDSCs were stimulated with BCG-GFP at 1 MOI for 16 h. Cytospins were then stained for Cav-1 and DAPI and analyzed by confocal microcopy. G-MDSC and M-MDSCs were defined on the basis of their nuclear shape. Scale bars upper row 7 μm, all other rows 10 μm. (F) Pearson's correlation coefficients for colocalization (“overlap”) of BCG and Cav-1 from n = 5 independent experiments like shown in (E) . Data are from **** P

    Techniques Used: Inhibition, Incubation, Flow Cytometry, Cytometry, Fluorescence, Mouse Assay, Staining

    56) Product Images from "Lipid profiles of prostate cancer cells"

    Article Title: Lipid profiles of prostate cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26222

    Distribution of lipids in prostate cancer cells (a-l) Micrographs of cross-sections through prostate cells that show the intracellular location of neutral and polar lipids. Cholesterol was depicted by staining cells with Filipin III (a-d). Neutral lipids such as triglycerides and cholesteryl esters were detected by staining cells with BODIPY ® 493/503 (e-h). ReZolve-L1™ (i-l) was used for staining polar lipids. Representative images from non-malignant PNT1a (a, e, i) and prostate cancer DU145 (b, f, j), 22RV1 (c, g, k) and LNCaP (d, h, l) cell lines. Prostate cells were fixed with 4% PFA (a-h) or imaged live (i-l). Scale bars, 20 μm.
    Figure Legend Snippet: Distribution of lipids in prostate cancer cells (a-l) Micrographs of cross-sections through prostate cells that show the intracellular location of neutral and polar lipids. Cholesterol was depicted by staining cells with Filipin III (a-d). Neutral lipids such as triglycerides and cholesteryl esters were detected by staining cells with BODIPY ® 493/503 (e-h). ReZolve-L1™ (i-l) was used for staining polar lipids. Representative images from non-malignant PNT1a (a, e, i) and prostate cancer DU145 (b, f, j), 22RV1 (c, g, k) and LNCaP (d, h, l) cell lines. Prostate cells were fixed with 4% PFA (a-h) or imaged live (i-l). Scale bars, 20 μm.

    Techniques Used: Staining

    57) Product Images from "Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin ?L?2 *"

    Article Title: Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin ?L?2 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.361519

    Murine BMDMs rolling on P-selectin use the same signaling cascade as murine bone marrow leukocytes to trigger β 2 integrin-dependent slow rolling on ICAM-1. A, velocities of WT BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of the indicated mAb. B, velocities of WT BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of the vehicle control DMSO, methyl-β-cyclodextrin ( M β CD ) or its inactive analog α-cyclodextrin (α CD ), MβCD plus 15% serum (to restore membrane cholesterol), or filipin III. C, velocities of WT or Hck −/− /Fgr −/− /Lyn −/− BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of the vehicle control DMSO, the Syk inhibitor piceatannol, the Src family kinase inhibitor PP2, or its inactive analog PP3. D, velocities of WT BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence of the vehicle control DMSO or the p38 inhibitor SB202190. E, velocities of WT, Btk −/− , or FcRγ −/− /DAP12 −/− BMDMs rolling on P-selectin with or without coimmobilized ICAM-1. The wall shear stress in A–E was 1 dyn/cm 2 . The data in A–F represent the mean ± S.E. from at least three experiments. *, p
    Figure Legend Snippet: Murine BMDMs rolling on P-selectin use the same signaling cascade as murine bone marrow leukocytes to trigger β 2 integrin-dependent slow rolling on ICAM-1. A, velocities of WT BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of the indicated mAb. B, velocities of WT BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of the vehicle control DMSO, methyl-β-cyclodextrin ( M β CD ) or its inactive analog α-cyclodextrin (α CD ), MβCD plus 15% serum (to restore membrane cholesterol), or filipin III. C, velocities of WT or Hck −/− /Fgr −/− /Lyn −/− BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence or absence of the vehicle control DMSO, the Syk inhibitor piceatannol, the Src family kinase inhibitor PP2, or its inactive analog PP3. D, velocities of WT BMDMs rolling on P-selectin with or without coimmobilized ICAM-1 in the presence of the vehicle control DMSO or the p38 inhibitor SB202190. E, velocities of WT, Btk −/− , or FcRγ −/− /DAP12 −/− BMDMs rolling on P-selectin with or without coimmobilized ICAM-1. The wall shear stress in A–E was 1 dyn/cm 2 . The data in A–F represent the mean ± S.E. from at least three experiments. *, p

    Techniques Used:

    58) Product Images from "Cell geometry dependent changes in plasma membrane order direct stem cell signalling and fate"

    Article Title: Cell geometry dependent changes in plasma membrane order direct stem cell signalling and fate

    Journal: Nature materials

    doi: 10.1038/s41563-017-0014-0

    Signal intensity of lipid raft markers is dependent on cell geometry. (a) Representative TIRF microscopy images of the plasma membrane-substrate interface. Cholera Toxin Subunit B (CTB) is a marker for lipid rafts and Filipin III stains for accumulation of cholesterol, a hallmark of lipid rafts. Caveolin-1 positive plasma membrane domains are a subtype of lipid rafts. Scale bars, 20 µm. (b) Quantification of TIRF images per condition and stain (n = 75–93 for CAV-1, n = 33–37 for filipin III, n = 38–53 for CTB). Box plots show complete data range, bottom and top of box represent 25% and 75%, respectively. ***, p
    Figure Legend Snippet: Signal intensity of lipid raft markers is dependent on cell geometry. (a) Representative TIRF microscopy images of the plasma membrane-substrate interface. Cholera Toxin Subunit B (CTB) is a marker for lipid rafts and Filipin III stains for accumulation of cholesterol, a hallmark of lipid rafts. Caveolin-1 positive plasma membrane domains are a subtype of lipid rafts. Scale bars, 20 µm. (b) Quantification of TIRF images per condition and stain (n = 75–93 for CAV-1, n = 33–37 for filipin III, n = 38–53 for CTB). Box plots show complete data range, bottom and top of box represent 25% and 75%, respectively. ***, p

    Techniques Used: Microscopy, CtB Assay, Marker, Staining

    59) Product Images from "Statins Impair Antitumor Effects of Rituximab by Inducing Conformational Changes of CD20The Potential Effect of Statins on Rituximab Immunotherapy"

    Article Title: Statins Impair Antitumor Effects of Rituximab by Inducing Conformational Changes of CD20The Potential Effect of Statins on Rituximab Immunotherapy

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0050064

    The Influence of Cholesterol-Modulating Drugs on the Expression of CD20 in Raji Cells (A–C) Raji cells were incubated with either diluents or MβCD (0–10 mg/ml) for 30 min, filipin III (0–1 μg/ml) for 30 min, or berberine (0–30 μg/ml) for 24 h. Then, equal numbers of cells (1 × 10 5 /well) were incubated for 60 min with 10 μg/ml rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured in a MTT assay. (D–F) Raji cells were incubated with either diluents or MβCD (5 mg/ml) for 30 min, filipin III (0.5 μg/ml) for 30 min, or berberine (25 μg/ml) for 24 h. Then, 1 × 10 6 /ml cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Control indicates the cells not incubated with MβCD, filipin III, and berberine, respectively. * p
    Figure Legend Snippet: The Influence of Cholesterol-Modulating Drugs on the Expression of CD20 in Raji Cells (A–C) Raji cells were incubated with either diluents or MβCD (0–10 mg/ml) for 30 min, filipin III (0–1 μg/ml) for 30 min, or berberine (0–30 μg/ml) for 24 h. Then, equal numbers of cells (1 × 10 5 /well) were incubated for 60 min with 10 μg/ml rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured in a MTT assay. (D–F) Raji cells were incubated with either diluents or MβCD (5 mg/ml) for 30 min, filipin III (0.5 μg/ml) for 30 min, or berberine (25 μg/ml) for 24 h. Then, 1 × 10 6 /ml cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Control indicates the cells not incubated with MβCD, filipin III, and berberine, respectively. * p

    Techniques Used: Expressing, Incubation, MTT Assay

    60) Product Images from "Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway"

    Article Title: Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway

    Journal:

    doi: 10.1111/j.1365-2567.2005.02304.x

    Uptake of IntSIIN. DC were incubated for 45 min with inhibitors (a) sodium azide/2-deoxyglucose (10, 1 m m ), (b) cytochalasin D (10, 1 μg/ml), (c) amiloride (6, 0·6 μ m ), (d) filipin III (10, 1 μg/ml), (e) nystatin (50, 10
    Figure Legend Snippet: Uptake of IntSIIN. DC were incubated for 45 min with inhibitors (a) sodium azide/2-deoxyglucose (10, 1 m m ), (b) cytochalasin D (10, 1 μg/ml), (c) amiloride (6, 0·6 μ m ), (d) filipin III (10, 1 μg/ml), (e) nystatin (50, 10

    Techniques Used: Incubation

    61) Product Images from "P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance"

    Article Title: P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1402845

    Clathrin- and caveolae/lipid raft–dependent endocytosis pathways are involved in LL-37 internalization by dTHP-1 cells. HMDMs ( n = 4) ( A ) or dTHP-1 cells ( n = 3) ( B ) were pretreated at 37°C for 1 h with the inhibitors of CME (CLP [10 μM], CLQ [10 μM], or dynasore [20 μM]) or the inhibitors of caveolae/lipid raft–dependent endocytosis (nystatin [10 μg/ml] or filipin [10 μM]). Then the cells were incubated with 10 μg/ml of FAM-labeled LL-37 for an additional hour, and MFI of the cells was analyzed by flow cytometry. ( C ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), CLP (10 μM), or CLQ (25 μM) at 37°C for 1 h and then challenged with LL-37 (10 μg/ml) for an additional hour. After three washes with PBS, the cells were lysed in RIPA buffer. LL-37 in cell lysates was detected by Western blots. The blot is one representative of three independent experiments ( upper panel ). Densitometric analysis of Western blots from three experiments ( lower panel ). ( D ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), dynasore (25 μM), or both inhibitors for 1 h. Subsequently, the cells were challenged with FAM–LL-37 (10 μg/ml), and MFI was analyzed by flow cytometry ( n = 3). ( E ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and CT-B, caveolin-1, or clathrin was visualized using a confocal microscope. ( F ) dTHP-1 cells were treated with TAMRA- or FAM-labeled LL-37 at 37°C for 1 h, and the colocalization of LL-37 and endosome, lysosome, or Golgi was visualized using a confocal microscope. The confocal images are representative of at least three experiments. Images in (E a )–(E c ) and (F a )–(F c ) are enlargements of the boxed areas. Scale bars, 10 μm. * p
    Figure Legend Snippet: Clathrin- and caveolae/lipid raft–dependent endocytosis pathways are involved in LL-37 internalization by dTHP-1 cells. HMDMs ( n = 4) ( A ) or dTHP-1 cells ( n = 3) ( B ) were pretreated at 37°C for 1 h with the inhibitors of CME (CLP [10 μM], CLQ [10 μM], or dynasore [20 μM]) or the inhibitors of caveolae/lipid raft–dependent endocytosis (nystatin [10 μg/ml] or filipin [10 μM]). Then the cells were incubated with 10 μg/ml of FAM-labeled LL-37 for an additional hour, and MFI of the cells was analyzed by flow cytometry. ( C ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), CLP (10 μM), or CLQ (25 μM) at 37°C for 1 h and then challenged with LL-37 (10 μg/ml) for an additional hour. After three washes with PBS, the cells were lysed in RIPA buffer. LL-37 in cell lysates was detected by Western blots. The blot is one representative of three independent experiments ( upper panel ). Densitometric analysis of Western blots from three experiments ( lower panel ). ( D ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), dynasore (25 μM), or both inhibitors for 1 h. Subsequently, the cells were challenged with FAM–LL-37 (10 μg/ml), and MFI was analyzed by flow cytometry ( n = 3). ( E ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and CT-B, caveolin-1, or clathrin was visualized using a confocal microscope. ( F ) dTHP-1 cells were treated with TAMRA- or FAM-labeled LL-37 at 37°C for 1 h, and the colocalization of LL-37 and endosome, lysosome, or Golgi was visualized using a confocal microscope. The confocal images are representative of at least three experiments. Images in (E a )–(E c ) and (F a )–(F c ) are enlargements of the boxed areas. Scale bars, 10 μm. * p

    Techniques Used: Incubation, Labeling, Flow Cytometry, Cytometry, Western Blot, Microscopy

    62) Product Images from "IFITM Proteins Inhibit Entry Driven by the MERS-Coronavirus Spike Protein: Evidence for Cholesterol-Independent Mechanisms"

    Article Title: IFITM Proteins Inhibit Entry Driven by the MERS-Coronavirus Spike Protein: Evidence for Cholesterol-Independent Mechanisms

    Journal: Viruses

    doi: 10.3390/v6093683

    Cholesterol trafficking inhibitors induce accumulation of intracellular cholesterol. Cos7 cells were treated with either DMSO (mock) or the indicated inhibitors for 21 h at a concentration of 10 µM (terconazole, clomiphene) or 2.5 µM (U18666A). Cells were then fixed, stained with filipin III (blue) and staining analyzed by fluorescence microscopy using equal exposure times.
    Figure Legend Snippet: Cholesterol trafficking inhibitors induce accumulation of intracellular cholesterol. Cos7 cells were treated with either DMSO (mock) or the indicated inhibitors for 21 h at a concentration of 10 µM (terconazole, clomiphene) or 2.5 µM (U18666A). Cells were then fixed, stained with filipin III (blue) and staining analyzed by fluorescence microscopy using equal exposure times.

    Techniques Used: Concentration Assay, Staining, Fluorescence, Microscopy

    63) Product Images from "Association of Herpesvirus Saimiri Tip with Lipid Raft Is Essential for Downregulation of T-Cell Receptor and CD4 Coreceptor"

    Article Title: Association of Herpesvirus Saimiri Tip with Lipid Raft Is Essential for Downregulation of T-Cell Receptor and CD4 Coreceptor

    Journal:

    doi: 10.1128/JVI.80.1.108-118.2006

    Recovery of CD3 and CD4 surface expression on Jurkat-Tip cells by Filipin III treatment. Jurkat-vector and Jurkat-Tip cells were treated with Filipin III (5 μg/ml) for the indicated times, and the surface expression of CD3, CD4, and CD45 was analyzed
    Figure Legend Snippet: Recovery of CD3 and CD4 surface expression on Jurkat-Tip cells by Filipin III treatment. Jurkat-vector and Jurkat-Tip cells were treated with Filipin III (5 μg/ml) for the indicated times, and the surface expression of CD3, CD4, and CD45 was analyzed

    Techniques Used: Expressing, Plasmid Preparation

    64) Product Images from "Lipid profiles of prostate cancer cells"

    Article Title: Lipid profiles of prostate cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26222

    Distribution of lipids in prostate cancer cells (a-l) Micrographs of cross-sections through prostate cells that show the intracellular location of neutral and polar lipids. Cholesterol was depicted by staining cells with Filipin III (a-d). Neutral lipids such as triglycerides and cholesteryl esters were detected by staining cells with BODIPY ® 493/503 (e-h). ReZolve-L1™ (i-l) was used for staining polar lipids. Representative images from non-malignant PNT1a (a, e, i) and prostate cancer DU145 (b, f, j), 22RV1 (c, g, k) and LNCaP (d, h, l) cell lines. Prostate cells were fixed with 4% PFA (a-h) or imaged live (i-l). Scale bars, 20 μm.
    Figure Legend Snippet: Distribution of lipids in prostate cancer cells (a-l) Micrographs of cross-sections through prostate cells that show the intracellular location of neutral and polar lipids. Cholesterol was depicted by staining cells with Filipin III (a-d). Neutral lipids such as triglycerides and cholesteryl esters were detected by staining cells with BODIPY ® 493/503 (e-h). ReZolve-L1™ (i-l) was used for staining polar lipids. Representative images from non-malignant PNT1a (a, e, i) and prostate cancer DU145 (b, f, j), 22RV1 (c, g, k) and LNCaP (d, h, l) cell lines. Prostate cells were fixed with 4% PFA (a-h) or imaged live (i-l). Scale bars, 20 μm.

    Techniques Used: Staining

    65) 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

    66) Product Images from "The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization"

    Article Title: The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization

    Journal: Molecules

    doi: 10.3390/molecules23123301

    TXA1.HCl treatment affects cholesterol localization in vitro of NCI-H460 cells. NCI-H460 cells were treated with TXA1.HCl (6.9 µM and 9.7 µM) for 24 and 48 h, or with respective controls. Cholesterol localization was evaluated by filipin staining. Images are representative of three independent experiments. Bar = 20 µM.
    Figure Legend Snippet: TXA1.HCl treatment affects cholesterol localization in vitro of NCI-H460 cells. NCI-H460 cells were treated with TXA1.HCl (6.9 µM and 9.7 µM) for 24 and 48 h, or with respective controls. Cholesterol localization was evaluated by filipin staining. Images are representative of three independent experiments. Bar = 20 µM.

    Techniques Used: In Vitro, Staining

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

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11073

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

    Techniques Used: Incubation, SDS Page, Marker

    68) Product Images from "Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze"

    Article Title: Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-07-0459

    Mitotic and interphase ER can be broken down by using filipin III. Cells were monitored at room temperature (≈20°C) after the addition of filipin III (5 μg/ml) at time points indicated at the top. (A) Metaphase cell. (B) Interphase cell. (C) Metaphase cell, possessing an artificial ER-Golgi hybrid, obtained by addition of BFA before M-phase entry (3 h before the first picture). (D) Interphase cell, likewise treated with BFA for 3 h. Bar, 10 μm.
    Figure Legend Snippet: Mitotic and interphase ER can be broken down by using filipin III. Cells were monitored at room temperature (≈20°C) after the addition of filipin III (5 μg/ml) at time points indicated at the top. (A) Metaphase cell. (B) Interphase cell. (C) Metaphase cell, possessing an artificial ER-Golgi hybrid, obtained by addition of BFA before M-phase entry (3 h before the first picture). (D) Interphase cell, likewise treated with BFA for 3 h. Bar, 10 μm.

    Techniques Used:

    Breakdown of the mitotic ER blocks diffusion of the ER but not the Golgi marker. Metaphase cells were treated with 5 μg/ml filipin III (open diamonds) or left as untreated controls (filled diamonds) for 30-45 min at room temperature, and then subjected to quantitative FRAP (at room temperature). Means ± SD, n = 10. (A) Golgi marker, GalNAc-T2-YFP. (B) ER marker, CFPSec61β. (C) Golgi marker GalNAc-T2-YFP, relocalized to the ER by addition of BFA before M-phase entry (3 h before addition of filipin III). Shown at right are cells representative of each curve (time points at bottom). Boxes indicate bleaching areas (2.5 × 2.5 μm).
    Figure Legend Snippet: Breakdown of the mitotic ER blocks diffusion of the ER but not the Golgi marker. Metaphase cells were treated with 5 μg/ml filipin III (open diamonds) or left as untreated controls (filled diamonds) for 30-45 min at room temperature, and then subjected to quantitative FRAP (at room temperature). Means ± SD, n = 10. (A) Golgi marker, GalNAc-T2-YFP. (B) ER marker, CFPSec61β. (C) Golgi marker GalNAc-T2-YFP, relocalized to the ER by addition of BFA before M-phase entry (3 h before addition of filipin III). Shown at right are cells representative of each curve (time points at bottom). Boxes indicate bleaching areas (2.5 × 2.5 μm).

    Techniques Used: Diffusion-based Assay, Marker

    69) Product Images from "Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage [S]"

    Article Title: Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.P013524

    Automated image analysis reliably quantifies cholesterol levels in Niemann Pick Type C (NPC) patient fibroblasts. (A) Primary human skin fibroblasts from an unaffected individual ( neg. ctrl 1 ; left panel), one NPC patient with moderately elevated cellular cholesterol (“variant” biochemical NPC phenotype; ΔNPC1_v03 ; middle panel), and one NPC patient with pronounced cholesterol storage (“classic” NPC-phenotype; ΔNPCx_c01 ; right panel). Cells were cultured under control conditions on glass-bottom slides, fixed, and stained with the cholesterol binding dye filipin. Images were acquired on an automated epifluorescence microscope. Bar = 20 μm. Perinuclear areas encompassing lysosomes (blue/red) were determined from microscopic images with masks generated by the automated image analysis software DetecTiff © . Green lines delineate extra-perinuclear background areas. (B) Scattergraph showing the correlation of mean perinuclear filipin signal intensity/cell within 11 selected images from four different fibroblast cultures, as quantified by DetecTiff © ( x axis) relative to manual quantification within masks generated with ImageJ software. On average, 71 cells/image were analyzed. (C) Scattergraph showing the correlation of mean perinuclear filipin signal intensity/cell from five different fibroblast cultures as quantified by DetecTiff © ( y axis; means from three to five images/cell line) relative to total cellular levels of free cholesterol as determined biochemically from cell extracts.
    Figure Legend Snippet: Automated image analysis reliably quantifies cholesterol levels in Niemann Pick Type C (NPC) patient fibroblasts. (A) Primary human skin fibroblasts from an unaffected individual ( neg. ctrl 1 ; left panel), one NPC patient with moderately elevated cellular cholesterol (“variant” biochemical NPC phenotype; ΔNPC1_v03 ; middle panel), and one NPC patient with pronounced cholesterol storage (“classic” NPC-phenotype; ΔNPCx_c01 ; right panel). Cells were cultured under control conditions on glass-bottom slides, fixed, and stained with the cholesterol binding dye filipin. Images were acquired on an automated epifluorescence microscope. Bar = 20 μm. Perinuclear areas encompassing lysosomes (blue/red) were determined from microscopic images with masks generated by the automated image analysis software DetecTiff © . Green lines delineate extra-perinuclear background areas. (B) Scattergraph showing the correlation of mean perinuclear filipin signal intensity/cell within 11 selected images from four different fibroblast cultures, as quantified by DetecTiff © ( x axis) relative to manual quantification within masks generated with ImageJ software. On average, 71 cells/image were analyzed. (C) Scattergraph showing the correlation of mean perinuclear filipin signal intensity/cell from five different fibroblast cultures as quantified by DetecTiff © ( y axis; means from three to five images/cell line) relative to total cellular levels of free cholesterol as determined biochemically from cell extracts.

    Techniques Used: Cell Culture, Staining, Binding Assay, Microscopy, Generated, Software

    70) Product Images from "Tumor-Specific Hsp70 Plasma Membrane Localization Is Enabled by the Glycosphingolipid Gb3"

    Article Title: Tumor-Specific Hsp70 Plasma Membrane Localization Is Enabled by the Glycosphingolipid Gb3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001925

    Hsp70 is associated with detergent resistant microdomains (DRMs). (A) Definition of the sublethal concentration of the raft disrupting agent methyl-beta cyclodextrin (MbCD). Following incubation of CX+ and CX− tumor sublines with 1 and 10 mM MbCD for 1 h, cells were washed and viability was determined by trypan blue staining after a 12 h recovery period. (B) The images show filipin III binding to untreated (ctrl, left graphs) and to CX+/CX− tumor sublines treated with the sublethal concentration of 1 mM MbCD (right graphs) for 1 h. (C) Cell surface density of Hsp70 in untreated (ctrl) and MbCD (1 mM, 10 mM) treated CX+ cells. Viability was 97.7±0.4 for control and 95±0.7 for MbCD treated cells. The data represent mean fluorescence intensity (mfi) values of three independent flow cytometric analyses. *Indicates values that are significantly different from control (P = 0.006).
    Figure Legend Snippet: Hsp70 is associated with detergent resistant microdomains (DRMs). (A) Definition of the sublethal concentration of the raft disrupting agent methyl-beta cyclodextrin (MbCD). Following incubation of CX+ and CX− tumor sublines with 1 and 10 mM MbCD for 1 h, cells were washed and viability was determined by trypan blue staining after a 12 h recovery period. (B) The images show filipin III binding to untreated (ctrl, left graphs) and to CX+/CX− tumor sublines treated with the sublethal concentration of 1 mM MbCD (right graphs) for 1 h. (C) Cell surface density of Hsp70 in untreated (ctrl) and MbCD (1 mM, 10 mM) treated CX+ cells. Viability was 97.7±0.4 for control and 95±0.7 for MbCD treated cells. The data represent mean fluorescence intensity (mfi) values of three independent flow cytometric analyses. *Indicates values that are significantly different from control (P = 0.006).

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

    71) Product Images from "Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development"

    Article Title: Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-07-0531

    UIS4 is required for maximal host LE and cholesterol recruitment to developing liver-stage P. berghei parasites. (A–C) Huh7 cells infected with GFP-expressing wild-type (WT), uis4 − , or ibis1 − parasites were fixed 24 h postinfection and stained with filipin (blue) and antibodies against CD63 (red) and GFP (green). Scale bars, 10 μm. (D) Huh7 cells were infected with P. berghei WT or mutant sporozoites, and infection was left to proceed until the indicated time points. Samples were stained with anti-GFP (parasite) and either anti-CD63 (LE/lysosomes; (top), anti-LAMP2 (LE/lysosomes; middle), or anti-LC3 (autophagic bodies; bottom). (E) WT and Atg5 −/− MEFs infected with P. berghei sporozoites were fixed 24 h postinfection and stained with antibodies against Pb Hsp70 (parasite) and LAMP2 (LE/lysosomes). At least 50 parasites per well were scored for host marker recruitment. Results are shown as a mean percentage of total parasites analyzed per well (±SD). One representative experiment of three performed is shown. ns, not significant; * p
    Figure Legend Snippet: UIS4 is required for maximal host LE and cholesterol recruitment to developing liver-stage P. berghei parasites. (A–C) Huh7 cells infected with GFP-expressing wild-type (WT), uis4 − , or ibis1 − parasites were fixed 24 h postinfection and stained with filipin (blue) and antibodies against CD63 (red) and GFP (green). Scale bars, 10 μm. (D) Huh7 cells were infected with P. berghei WT or mutant sporozoites, and infection was left to proceed until the indicated time points. Samples were stained with anti-GFP (parasite) and either anti-CD63 (LE/lysosomes; (top), anti-LAMP2 (LE/lysosomes; middle), or anti-LC3 (autophagic bodies; bottom). (E) WT and Atg5 −/− MEFs infected with P. berghei sporozoites were fixed 24 h postinfection and stained with antibodies against Pb Hsp70 (parasite) and LAMP2 (LE/lysosomes). At least 50 parasites per well were scored for host marker recruitment. Results are shown as a mean percentage of total parasites analyzed per well (±SD). One representative experiment of three performed is shown. ns, not significant; * p

    Techniques Used: Infection, Expressing, Staining, Mutagenesis, Marker

    Release of cholesterol from LE/lysosomes rescues P. berghei development in U18666A-treated cells. Huh7 cells were treated with 3 μM U18666A or 1 mM MβCD 36 h before infection. Where indicated, MβCD was added to U18666A-treated cells 12 h before infection. Cells were either fixed and stained with filipin (A; scale bars, 30 μm) or infected with P. berghei sporozoites and kept under continual presence of the drug until fixation 24 h postinfection (B). Parasites were visualized by staining with an anti- Pb Hsp70 antibody. Size was assessed, and the area of each measured parasite was plotted individually. One representative of three independent experiments is shown. * p
    Figure Legend Snippet: Release of cholesterol from LE/lysosomes rescues P. berghei development in U18666A-treated cells. Huh7 cells were treated with 3 μM U18666A or 1 mM MβCD 36 h before infection. Where indicated, MβCD was added to U18666A-treated cells 12 h before infection. Cells were either fixed and stained with filipin (A; scale bars, 30 μm) or infected with P. berghei sporozoites and kept under continual presence of the drug until fixation 24 h postinfection (B). Parasites were visualized by staining with an anti- Pb Hsp70 antibody. Size was assessed, and the area of each measured parasite was plotted individually. One representative of three independent experiments is shown. * p

    Techniques Used: Infection, Staining

    72) Product Images from "The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells"

    Article Title: The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192250

    Localization of Slp3p. (A) Exponential-phase samples of the untagged wt SLP3 control strain and (B) SLP3-YFP expressing strain were treated with 160 μM FM 4–64. The FM 4–64 incubation period was set to allow visualization of the vacuole. Cells were viewed under bright-field (BF) and fluorescent microscopy. The right panels show an overlay of the YFP and FM 4–64 fluorescence (YFP/FM 4–64). (C) Overlay photo shown in (B) was recolored with ImageJ to assess Slp3p-Yfp vacuolar membrane localization. The yellow fluorescence color was reassigned to green. (D) Overnight-cultured SLP3-YFP cells were treated with 200 μg/mL Filipin and viewed using fluorescent microscopy. Image depicts a single cell, and the right panel shows an overlay of the YFP and Filipin fluorescence (YFP/Filipin). The dashed white box highlights the cell depicted in the inset image. For each assay, three biological replicates were analyzed. Experiments were repeated at least three times, and data presented represents one representative experiment. Approximately 1.0 x 10 4 cells of each strain were selected for viewing (A-D). Scale bars represent 10 μm.
    Figure Legend Snippet: Localization of Slp3p. (A) Exponential-phase samples of the untagged wt SLP3 control strain and (B) SLP3-YFP expressing strain were treated with 160 μM FM 4–64. The FM 4–64 incubation period was set to allow visualization of the vacuole. Cells were viewed under bright-field (BF) and fluorescent microscopy. The right panels show an overlay of the YFP and FM 4–64 fluorescence (YFP/FM 4–64). (C) Overlay photo shown in (B) was recolored with ImageJ to assess Slp3p-Yfp vacuolar membrane localization. The yellow fluorescence color was reassigned to green. (D) Overnight-cultured SLP3-YFP cells were treated with 200 μg/mL Filipin and viewed using fluorescent microscopy. Image depicts a single cell, and the right panel shows an overlay of the YFP and Filipin fluorescence (YFP/Filipin). The dashed white box highlights the cell depicted in the inset image. For each assay, three biological replicates were analyzed. Experiments were repeated at least three times, and data presented represents one representative experiment. Approximately 1.0 x 10 4 cells of each strain were selected for viewing (A-D). Scale bars represent 10 μm.

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

    73) Product Images from "The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization"

    Article Title: The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization

    Journal: Molecules

    doi: 10.3390/molecules23123301

    TXA1.HCl treatment affects cholesterol localization in vitro of NCI-H460 cells. NCI-H460 cells were treated with TXA1.HCl (6.9 µM and 9.7 µM) for 24 and 48 h, or with respective controls. Cholesterol localization was evaluated by filipin staining. Images are representative of three independent experiments. Bar = 20 µM.
    Figure Legend Snippet: TXA1.HCl treatment affects cholesterol localization in vitro of NCI-H460 cells. NCI-H460 cells were treated with TXA1.HCl (6.9 µM and 9.7 µM) for 24 and 48 h, or with respective controls. Cholesterol localization was evaluated by filipin staining. Images are representative of three independent experiments. Bar = 20 µM.

    Techniques Used: In Vitro, Staining

    74) Product Images from "The in vitro characterization of polyene glycosyltransferases AmphDI and NysDI"

    Article Title: The in vitro characterization of polyene glycosyltransferases AmphDI and NysDI

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.200800349

    Naturally-occurring polyene macrolides Amphotericin B ( 1 ), nystatin A1 ( 2 ), candicidin/FR-008 ( 3 ), pimaricin ( 4 ), rimocidin ( 5 ) and filipin III ( 6 ) are all produced by Streptomyces strains and genetic loci for 1–5 have been characterized. The corresponding mycosaminyltransferases (AmphDI, NysDI, FscMI, PimK and RimE, respectively) responsible for glycoside formation are highlighted.
    Figure Legend Snippet: Naturally-occurring polyene macrolides Amphotericin B ( 1 ), nystatin A1 ( 2 ), candicidin/FR-008 ( 3 ), pimaricin ( 4 ), rimocidin ( 5 ) and filipin III ( 6 ) are all produced by Streptomyces strains and genetic loci for 1–5 have been characterized. The corresponding mycosaminyltransferases (AmphDI, NysDI, FscMI, PimK and RimE, respectively) responsible for glycoside formation are highlighted.

    Techniques Used: Produced

    75) Product Images from "The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis"

    Article Title: The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045028

    Treatment of epithelial cells with Filipin III disrupts mycobacterial-induced LR aggregation. A549 cells were treated with cholesterol-binding, LR-disruption agent Filipin III and LR aggregation observed for controls and infections with all three live Mtb strains. Confocal microscopy demonstrated an absence of CT-B puncta at 6 and 24 hpi for controls (A) and live Mtb strains (B). Images were collected at 63x magnification. Infections were performed in triplicate and repeated three times.
    Figure Legend Snippet: Treatment of epithelial cells with Filipin III disrupts mycobacterial-induced LR aggregation. A549 cells were treated with cholesterol-binding, LR-disruption agent Filipin III and LR aggregation observed for controls and infections with all three live Mtb strains. Confocal microscopy demonstrated an absence of CT-B puncta at 6 and 24 hpi for controls (A) and live Mtb strains (B). Images were collected at 63x magnification. Infections were performed in triplicate and repeated three times.

    Techniques Used: Binding Assay, Confocal Microscopy

    Related Articles

    Expressing:

    Article Title: Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors
    Article Snippet: Doxycycline hyclate (DOX) (D9891, Sigma-Aldrich, San Luis, MS, USA) for cell–cell fusion reporter expression activation was prepared as 50 mg/mL stock solution in DMSO and stored at −80 °C. .. Filipin III (F4767) for unesterified cholesterol detection was obtained from Sigma-Aldrich (San Luis, MS, USA).

    Synthesized:

    Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis
    Article Snippet: Dimethyl sulfoxide (D2650), Histopaque 1077, saponin, BSA (A9576), PitStop 2, phenylarsine oxide (PAO; P3075), monodansyl cadaverine (MDC; 30432), methyl-β-cyclodextrin (C4555), Filipin (F4767), CK666, CK689, 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; D8417) and Brefeldin A (B5936) were purchased from Sigma Aldrich (St Louis, MO). .. LTB4 , and Y27632 were from Cayman Chemical. fMLF–Alexa-Fluor-488 was synthesized by Discovery Peptides, Cambridge Research Biochemicals.

    Cytometry:

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: Doxorubicin-release detection In this study, the doxorubicin-release detection was performed by flow cytometry and immunofluorescence. .. In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway).

    Blocking Assay:

    Article Title: PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux
    Article Snippet: For filipin–cholesterol labeling, mouse Schwann cells were fixed with 4% paraformaldehyde for 10 min. After rinsing in PBS, samples were incubated with 50 μg/ml filipin III (F4767; Sigma-Aldrich) in PBS for 1 h in the dark. .. For ABCA1 and PMP22 costaining, sciatic nerve sections were permeabilized with ice-cold methanol for 5 min. For ABCA1 staining on teased fibers and sectioned nerves, samples were fixed for 15–30 min with 4% PFA and permeabilized with 0.1% Triton X-100 for 15 min. After blocking in 15% normal goat serum for 1 h and incubating with primary antibodies overnight at 4°C, bound primary antibodies were detected with appropriate fluorescent-dye conjugated secondary antibodies (Invitrogen).

    Incubation:

    Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation
    Article Snippet: Fluorescence staining for lipids and unesterified cholesterol For lipid staining, cryo liver sections (5 μm; co, n = 10; Igf2, n = 11) were fixed with 4% normal buffered formalin for 5 min, washed three times for 10 min with PBS, incubated with the highly selective fluorescent lipid dye LD540 (Astanina et al., ) (0.5 μg/ml in PBS) for 15 min at room temperature, washed three times with PBS for 10 min, incubated for 1 min with diaminophenylindol HCl (DAPI) (D9542, Sigma Aldrich) at a concentration of 1 μg/ml, and afterwards washed three times with PBS. .. For the staining of free cholesterol, we performed filipin (F4767-1 mg, Sigma Aldrich) staining according to published methods (Ioannou et al., ; Simon et al., ).

    Article Title: Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex
    Article Snippet: .. After 24 hours, media was removed and then cells were washed 3 times in PBS and incubated with 50 µg/mL filipin (F4767 Sigma Aldrich, St. Louis, MO) in PBS for one hour in the dark at room temperature, followed by 3 washes with PBS. .. Images were acquired on a Carl Zeiss AxioObserver Z1 fluorescent wide field microscope.

    Article Title: SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20
    Article Snippet: .. The cells were then incubated with 50 μg/ml filipin (Sigma, F4767) (in PBS made up with 10% FBS) for 2 h at room temperature in the dark. .. When a nuclear counter stain was used for quantitation of nuclear-peripheral distribution, 5 μ m Syto® 85 orange fluorescent nucleic acid stain (Molecular Probes, S-11366) was included in the filipin staining solution.

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: .. For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps. .. In this case, the nuclei were stained with RedDot2 (catalog no. 40061; Biotium).

    Article Title: PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux
    Article Snippet: .. For filipin–cholesterol labeling, mouse Schwann cells were fixed with 4% paraformaldehyde for 10 min. After rinsing in PBS, samples were incubated with 50 μg/ml filipin III (F4767; Sigma-Aldrich) in PBS for 1 h in the dark. .. For filipin costaining with GS15, after 4% PFA fixation, cells were incubated with filipin (50 μg/ml) for 1 h before adding GS15 antibody.

    Mass Spectrometry:

    Article Title: Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors
    Article Snippet: .. Filipin III (F4767) for unesterified cholesterol detection was obtained from Sigma-Aldrich (San Luis, MS, USA). .. Mouse monoclonal allophycocyanin(APC)-conjugated antibodies (mAbs) against human surface entry factors Axl (FAB154A), human integrin β1 (FAB17783A), Mer (FAB8912A), or DC-SIGN (FAB161A) and their respective isotype controls, IgG1 (IC002A) and IgG2B (IC0041A) as well as AlexaFluor488-conjugated mAb against human integrin αV (FAB1219G-025) and its IgG1 isotype control (IC002G) were purchased from R & D systems (Minneapolis, MN, USA).

    Western Blot:

    Article Title: Uptake of Shiga-toxigenic Escherichia coli SubAB by HeLa cells requires an actin– and lipid raft -dependent pathway
    Article Snippet: Methyl-β-cyclodextrin (c45551G), Dynasore hydrate (D7693), Filipin III (F4767), ionomycin (I9657), cytochalasin D (C8273) and EGTA were from Sigma Aldrich; Chlorpromazine hydrochloride (ALX270171G001), 5-ethylisopropyl amiloride (ALX550266M005) and Rottlerin (ALX350075M010) from Enzo Life Science; and PI3K inhibitors, LY294002 (9901) and wortmannin (9951S) from Cell Signaling Technology. .. For Western blot analysis, anti-BiP/GRP78 (610978), anti-clathrin (610499), anti-caveolin I (610406), anti-caveolin II (610684) and anti-PKCδ (610398) antibodies were purchased from Becton Dickinson, and anti-dynamin I/II antibody (2342) was from Cell Signaling Technology.

    Flow Cytometry:

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: For flow cytometric analysis, the drug-loaded cells were washed with ice cold PBS twice and detected in the FL-2 channel depending on the red fluorescence of doxorubicin. .. In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway).

    Concentration Assay:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: .. To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml. .. The MagicRed CathepsinB Assay Kit (ImmunoChemistry Technologies, 937) was used following the manufacturer's instructions.

    Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation
    Article Snippet: Fluorescence staining for lipids and unesterified cholesterol For lipid staining, cryo liver sections (5 μm; co, n = 10; Igf2, n = 11) were fixed with 4% normal buffered formalin for 5 min, washed three times for 10 min with PBS, incubated with the highly selective fluorescent lipid dye LD540 (Astanina et al., ) (0.5 μg/ml in PBS) for 15 min at room temperature, washed three times with PBS for 10 min, incubated for 1 min with diaminophenylindol HCl (DAPI) (D9542, Sigma Aldrich) at a concentration of 1 μg/ml, and afterwards washed three times with PBS. .. For the staining of free cholesterol, we performed filipin (F4767-1 mg, Sigma Aldrich) staining according to published methods (Ioannou et al., ; Simon et al., ).

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: .. For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps. .. In this case, the nuclei were stained with RedDot2 (catalog no. 40061; Biotium).

    Protease Inhibitor:

    Article Title: Arv1 promotes cell division by recruiting IQGAP1 and myosin to the cleavage furrow
    Article Snippet: Protease Inhibitor Cocktail was provided by Roche. .. N-ethylmaleimide, Filipin III (F4767), Phosphatase Inhibitor Cocktail 2, Thymidine, MβCD and Nocodazole were from Sigma Aldrich.

    Immunolabeling:

    Article Title: PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux
    Article Snippet: Paragraph title: Fluorescent dye staining and immunolabeling. ... For filipin–cholesterol labeling, mouse Schwann cells were fixed with 4% paraformaldehyde for 10 min. After rinsing in PBS, samples were incubated with 50 μg/ml filipin III (F4767; Sigma-Aldrich) in PBS for 1 h in the dark.

    Cell Culture:

    Article Title: SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20
    Article Snippet: HEK293 cells were cultured on mouse Laminin (Invitrogen, 23017-015) coated plastic to prevent them from washing off during in later steps. .. The cells were then incubated with 50 μg/ml filipin (Sigma, F4767) (in PBS made up with 10% FBS) for 2 h at room temperature in the dark.

    Indirect Immunoperoxidase Assay:

    Article Title: Uptake of Shiga-toxigenic Escherichia coli SubAB by HeLa cells requires an actin– and lipid raft -dependent pathway
    Article Snippet: Methyl-β-cyclodextrin (c45551G), Dynasore hydrate (D7693), Filipin III (F4767), ionomycin (I9657), cytochalasin D (C8273) and EGTA were from Sigma Aldrich; Chlorpromazine hydrochloride (ALX270171G001), 5-ethylisopropyl amiloride (ALX550266M005) and Rottlerin (ALX350075M010) from Enzo Life Science; and PI3K inhibitors, LY294002 (9901) and wortmannin (9951S) from Cell Signaling Technology. .. PAK inhibitor IPA-3 and Bafilomycin A1 were obtained from WAKO.

    Inhibition:

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: .. In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway). .. For immunofluorescence staining, the drug-loaded cells were washed with ice cold PBS twice and stained with DAPI for nuclear staining after fixing with 3.7% paraformaldehyde for 15 minutes.

    Imaging:

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway). .. The fixed cells were washed with ice cold PBS twice, re-suspended in mounting solution, and dropped onto the cover slide for analysis by fluorescence microscopy (iRiS™ digital cell imaging system, Logos Biosystems, Inc., Seoul, South Korea).

    Article Title: SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20
    Article Snippet: The cells were then incubated with 50 μg/ml filipin (Sigma, F4767) (in PBS made up with 10% FBS) for 2 h at room temperature in the dark. .. After 2 h, the cells were washed three times with PBS and stored in the dark at 4°C before imaging using an Olympus IX71 microscope.

    Sequencing:

    Article Title: Ligand-Dependent Recruitment of the ErbB4 Signaling Complex into Neuronal Lipid Rafts
    Article Snippet: Methyl-β-cyclodextrin (MCD) (M1356) and filipin (F4767) were from Sigma. .. ErbB4 (aa 26–1308) was subcloned into Bam HI– Sal I sites of pFLAG-CMV downstream of an artificial signal peptide sequence and a FLAG epitope.

    Affinity Purification:

    Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice
    Article Snippet: We also used rabbit monoclonal antibodies against Lamp1 (D2D11, 9091, Cell Signaling, 1/500); and rabbit polyclonal antibody against EEA1 (ALX‐210‐239, ENZO life science, 1/400); as well as affinity‐purified donkey antibodies against mouse IgG, Alexa 488‐labelled (715,545,151, Jackson ImmunoResearch, 1/200) and against rabbit IgG, Cy3‐labelled (ref: 711,165,152, Jackson ImmunoResearch, 1/200). .. We obtained filipin (F4767) from Sigma; BODIPY‐493/503 Methyl Bromide (used at 1/1,000 from 1 mg/ml ethanol stock) and CellTracker green (C2925) from Thermo Fisher Scientific; and RNase (12091‐021) and propidium iodide from (Ref. P3566) from Invitrogen.

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: PI4P staining of the internal membranes and NS5A staining were performed, as previously described ( ) with the primary antibodies anti-PI4P IgM (1:300; catalog no. Z-P004; Echelon) or affinity-purified rabbit anti-NS5A antibody (1:2,000 [ ]), and secondary antibodies goat anti-mouse IgM or goat anti-rabbit IgG conjugated to Alexa Fluor 568 (catalog no. A-21042; Invitrogen) or Alexa Fluor 488 (catalog no. A-11011; Invitrogen) (1:600). .. For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps.

    Recombinant:

    Article Title: Ligand-Dependent Recruitment of the ErbB4 Signaling Complex into Neuronal Lipid Rafts
    Article Snippet: Methyl-β-cyclodextrin (MCD) (M1356) and filipin (F4767) were from Sigma. .. For NRG stimulation, we used a recombinant polypeptide containing the entire epidermal growth factor domain of the β-type NRG-1 (rHRGβ 177–244 ) , which binds to ErbB3 and ErbB4 and thus induces tyrosine phosphorylation of ErbB2, ErbB3, and ErbB4 ( ; ; ).

    Article Title: Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors
    Article Snippet: Recombinant conjugated protein EGF-AlexaFluor555 for EGF intravesicular accumulation analysis was purchased from Invitrogen™ (E35350) (Carlsbad, CA, USA). .. Filipin III (F4767) for unesterified cholesterol detection was obtained from Sigma-Aldrich (San Luis, MS, USA).

    Immunofluorescence:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: Paragraph title: Immunofluorescence microscopy ... To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml.

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: Doxorubicin-release detection In this study, the doxorubicin-release detection was performed by flow cytometry and immunofluorescence. .. In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway).

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: Paragraph title: Indirect immunofluorescence. ... For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps.

    Fluorescence:

    Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation
    Article Snippet: Paragraph title: Fluorescence staining for lipids and unesterified cholesterol ... For the staining of free cholesterol, we performed filipin (F4767-1 mg, Sigma Aldrich) staining according to published methods (Ioannou et al., ; Simon et al., ).

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: For flow cytometric analysis, the drug-loaded cells were washed with ice cold PBS twice and detected in the FL-2 channel depending on the red fluorescence of doxorubicin. .. In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway).

    Article Title: A Mouse Model of Schnyder Corneal Dystrophy with the N100S Point Mutation
    Article Snippet: Sections were stained at room temperature for 75 minutes with freshly made 0.05 mg/mL filipin III solution (Cat F4767, Sigma, St. Louis, MO). .. Confocal microscopic images of filipin fluorescence were obtained with a Zeiss 780 microscope and Plan-Apochromat 63 ×/1.40 oil objective using 355 nm wavelength for excitation and 371 to 501 nm wavelengths for fluorescence emission.

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps. .. The slides were then mounted with 5 μl of Dako fluorescence mounting medium (catalog no. S3023; Dako North America, Inc.), and images were captured using a Leica SP5 AOBS confocal microscope equipped with a Leica objective HCX PL APO 63×/1.4.

    Size-exclusion Chromatography:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: To reduce cytosolic background cells were extracted with 0.02% saponin (Sigma, F47036) for 30 sec before fixation. .. To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml.

    Labeling:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: .. To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml. .. The MagicRed CathepsinB Assay Kit (ImmunoChemistry Technologies, 937) was used following the manufacturer's instructions.

    Article Title: PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux
    Article Snippet: .. For filipin–cholesterol labeling, mouse Schwann cells were fixed with 4% paraformaldehyde for 10 min. After rinsing in PBS, samples were incubated with 50 μg/ml filipin III (F4767; Sigma-Aldrich) in PBS for 1 h in the dark. .. For filipin costaining with GS15, after 4% PFA fixation, cells were incubated with filipin (50 μg/ml) for 1 h before adding GS15 antibody.

    Microscopy:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: Paragraph title: Immunofluorescence microscopy ... To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml.

    Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation
    Article Snippet: For the staining of free cholesterol, we performed filipin (F4767-1 mg, Sigma Aldrich) staining according to published methods (Ioannou et al., ; Simon et al., ). .. Slides were embedded in the water based FluoroSafe™ mounting medium and visualized with an inverse fluorescence microscope at 558/583 (excitation/emission) for LD540 and at 359/461 (excitation/emission) for DAPI and filipin (Axio Observer, Zeiss, Feldbach, Swiss).

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway). .. The fixed cells were washed with ice cold PBS twice, re-suspended in mounting solution, and dropped onto the cover slide for analysis by fluorescence microscopy (iRiS™ digital cell imaging system, Logos Biosystems, Inc., Seoul, South Korea).

    Article Title: Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex
    Article Snippet: After 24 hours, media was removed and then cells were washed 3 times in PBS and incubated with 50 µg/mL filipin (F4767 Sigma Aldrich, St. Louis, MO) in PBS for one hour in the dark at room temperature, followed by 3 washes with PBS. .. Images were acquired on a Carl Zeiss AxioObserver Z1 fluorescent wide field microscope.

    Article Title: A Mouse Model of Schnyder Corneal Dystrophy with the N100S Point Mutation
    Article Snippet: Sections were stained at room temperature for 75 minutes with freshly made 0.05 mg/mL filipin III solution (Cat F4767, Sigma, St. Louis, MO). .. Confocal microscopic images of filipin fluorescence were obtained with a Zeiss 780 microscope and Plan-Apochromat 63 ×/1.40 oil objective using 355 nm wavelength for excitation and 371 to 501 nm wavelengths for fluorescence emission.

    Article Title: SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20
    Article Snippet: The cells were then incubated with 50 μg/ml filipin (Sigma, F4767) (in PBS made up with 10% FBS) for 2 h at room temperature in the dark. .. After 2 h, the cells were washed three times with PBS and stored in the dark at 4°C before imaging using an Olympus IX71 microscope.

    Article Title: Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice
    Article Snippet: We obtained filipin (F4767) from Sigma; BODIPY‐493/503 Methyl Bromide (used at 1/1,000 from 1 mg/ml ethanol stock) and CellTracker green (C2925) from Thermo Fisher Scientific; and RNase (12091‐021) and propidium iodide from (Ref. P3566) from Invitrogen. .. When indicated, the cells were treated with 10 μM thioperamide or DMSO (1/2000) for 72 h and imaged by automated microscope.

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps. .. The slides were then mounted with 5 μl of Dako fluorescence mounting medium (catalog no. S3023; Dako North America, Inc.), and images were captured using a Leica SP5 AOBS confocal microscope equipped with a Leica objective HCX PL APO 63×/1.4.

    Software:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml. .. To analyze colocalization, the Pearson's coefficient was calculated from confocal images using the Volocity 5.2 software (Perkin Elmer).

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps. .. We used the Volocity image processing software (Improvision, PerkinElmer) to quantify the intensity fluorescence of the images.

    Quantitation Assay:

    Article Title: SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20
    Article Snippet: The cells were then incubated with 50 μg/ml filipin (Sigma, F4767) (in PBS made up with 10% FBS) for 2 h at room temperature in the dark. .. When a nuclear counter stain was used for quantitation of nuclear-peripheral distribution, 5 μ m Syto® 85 orange fluorescent nucleic acid stain (Molecular Probes, S-11366) was included in the filipin staining solution.

    Activation Assay:

    Article Title: Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors
    Article Snippet: Doxycycline hyclate (DOX) (D9891, Sigma-Aldrich, San Luis, MS, USA) for cell–cell fusion reporter expression activation was prepared as 50 mg/mL stock solution in DMSO and stored at −80 °C. .. Filipin III (F4767) for unesterified cholesterol detection was obtained from Sigma-Aldrich (San Luis, MS, USA).

    FLAG-tag:

    Article Title: Ligand-Dependent Recruitment of the ErbB4 Signaling Complex into Neuronal Lipid Rafts
    Article Snippet: Methyl-β-cyclodextrin (MCD) (M1356) and filipin (F4767) were from Sigma. .. ErbB4 (aa 26–1308) was subcloned into Bam HI– Sal I sites of pFLAG-CMV downstream of an artificial signal peptide sequence and a FLAG epitope.

    Staining:

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion
    Article Snippet: F-actin was visualized using Alexa Fluor 568-coupled phalloidin (Sigma Aldrich, A12380) and cell nuclei were stained with Hoechst (Invitrogen, H3570). .. To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml.

    Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation
    Article Snippet: .. For the staining of free cholesterol, we performed filipin (F4767-1 mg, Sigma Aldrich) staining according to published methods (Ioannou et al., ; Simon et al., ). .. Slides were embedded in the water based FluoroSafe™ mounting medium and visualized with an inverse fluorescence microscope at 558/583 (excitation/emission) for LD540 and at 359/461 (excitation/emission) for DAPI and filipin (Axio Observer, Zeiss, Feldbach, Swiss).

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells
    Article Snippet: In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway). .. For immunofluorescence staining, the drug-loaded cells were washed with ice cold PBS twice and stained with DAPI for nuclear staining after fixing with 3.7% paraformaldehyde for 15 minutes.

    Article Title: A Mouse Model of Schnyder Corneal Dystrophy with the N100S Point Mutation
    Article Snippet: .. Sections were stained at room temperature for 75 minutes with freshly made 0.05 mg/mL filipin III solution (Cat F4767, Sigma, St. Louis, MO). .. Following staining, sections were mounted in ProLong™ Diamond Antifade mounting medium (Thermo Fisher Scientific, Waltham, MA).

    Article Title: SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20
    Article Snippet: The cells were then incubated with 50 μg/ml filipin (Sigma, F4767) (in PBS made up with 10% FBS) for 2 h at room temperature in the dark. .. When a nuclear counter stain was used for quantitation of nuclear-peripheral distribution, 5 μ m Syto® 85 orange fluorescent nucleic acid stain (Molecular Probes, S-11366) was included in the filipin staining solution.

    Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis
    Article Snippet: Dimethyl sulfoxide (D2650), Histopaque 1077, saponin, BSA (A9576), PitStop 2, phenylarsine oxide (PAO; P3075), monodansyl cadaverine (MDC; 30432), methyl-β-cyclodextrin (C4555), Filipin (F4767), CK666, CK689, 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; D8417) and Brefeldin A (B5936) were purchased from Sigma Aldrich (St Louis, MO). .. The formyl-Nle-Leu-Phe-Nle-Tyr-Lys fluorescein derivative (fNLFNYK–FITC), Rhodamine–phalloidin, Alexa Fluor 647-conjugated phalloidin, Alexa Fluor 568-conjugated transferrin from human serum, Alexa Fluor 555-conjugated Cholera toxin B subunit (CTxB), CellTracker Red CMPTX dye, CellMask Deep Red Plasma membrane stain and LysoTracker Red DND-99, Alexa Fluor 568-conjugated donkey antibodies to IgG (mouse, rabbit and goat) and Latrunculin A (LatA) were obtained from Molecular Probes.

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes
    Article Snippet: .. For staining free cholesterol, the cells were incubated with Filipin III (catalog no. F4767; Sigma-Aldrich) at a concentration of 0.2 mg/ml in Tris-buffered saline (TBS) for 45 min during the final washing steps. .. In this case, the nuclei were stained with RedDot2 (catalog no. 40061; Biotium).

    Article Title: PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux
    Article Snippet: Paragraph title: Fluorescent dye staining and immunolabeling. ... For filipin–cholesterol labeling, mouse Schwann cells were fixed with 4% paraformaldehyde for 10 min. After rinsing in PBS, samples were incubated with 50 μg/ml filipin III (F4767; Sigma-Aldrich) in PBS for 1 h in the dark.

    other:

    Article Title: Blebbishields, the emergency program for cancer stem cells: sphere formation and tumorigenesis after apoptosis
    Article Snippet: Lithium chloride (L-4408), Tunicamycin (T7765), Heparin (H3393), Filipin-III (F4767), Amiloride (A7410), Esomeprazole (E7906), and Lactic acid (L1875) were from Sigma (St Louis, MO, USA).

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    Kinetics and mechanism of reaction of S S diphenylsulfilimine with 1 fluoro 2 4 dinitrobenzene 1 chloro 2 4 dinitrobenzene 2 chloro 3 nitropyridine 2 chloro 5 nitropyridine and 2
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    95
    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: 95/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Overall structures of CYP105P1-filipin I complex ( A ) and unliganded CYP105D6 ( B ), illustrated by ribbon representation. Heme and ligands are shown as stick models. The BC and FG loop regions are shown in dark gray. a.a. , amino acids.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Overall structures of CYP105P1-filipin I complex ( A ) and unliganded CYP105D6 ( B ), illustrated by ribbon representation. Heme and ligands are shown as stick models. The BC and FG loop regions are shown in dark gray. a.a. , amino acids.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques:

    Spectral changes of CYP105P1 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 422 nm ( C ).

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Spectral changes of CYP105P1 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 422 nm ( C ).

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques: Titration

    Interactions between CYP105P1 and filipin I. A , shown is an F obs − F calc omit electron density map of the filipin I molecule contoured at 4.0 σ. B , hydrophobic interactions are observed at both sides of the 28-membered ring. Labels for atoms of filipin I are underlined. C , a stereographic figure shows interactions with the BC loop, FG helices, and I helix. The water molecules mediate hydrophilic interactions with the polyol group of filipin I. The extensive hydrogen-bonding network and residues involved in it are shown as dotted lines and stick models, respectively. The distance between the C26 atom of filipin I and the heme iron is 5.0 Å, and the pro-S hydrogen side is directed toward the heme.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Interactions between CYP105P1 and filipin I. A , shown is an F obs − F calc omit electron density map of the filipin I molecule contoured at 4.0 σ. B , hydrophobic interactions are observed at both sides of the 28-membered ring. Labels for atoms of filipin I are underlined. C , a stereographic figure shows interactions with the BC loop, FG helices, and I helix. The water molecules mediate hydrophilic interactions with the polyol group of filipin I. The extensive hydrogen-bonding network and residues involved in it are shown as dotted lines and stick models, respectively. The distance between the C26 atom of filipin I and the heme iron is 5.0 Å, and the pro-S hydrogen side is directed toward the heme.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques:

    Active site structures of CYP105P1-filipin I ( green ) superimposed with CYP105D6 ( A , cyan ), P450cam-camphor-O 2 ( B , yellow ), and P450 EryK-erythromycin D ( C , magenta ). Water molecules and the hydroxylation target positions of substrates (C26 of filipin I, C5 of camphor, and C12 of erythromycin D) are shown as spheres . A water molecule is positioned 2.8 Å from the heme iron in the CYP105D6 structure ( A ).

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Active site structures of CYP105P1-filipin I ( green ) superimposed with CYP105D6 ( A , cyan ), P450cam-camphor-O 2 ( B , yellow ), and P450 EryK-erythromycin D ( C , magenta ). Water molecules and the hydroxylation target positions of substrates (C26 of filipin I, C5 of camphor, and C12 of erythromycin D) are shown as spheres . A water molecule is positioned 2.8 Å from the heme iron in the CYP105D6 structure ( A ).

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques:

    Closing motion of CYP105P1. Shown is a stereographic superimposition of the filipin I complex structure with the ligand-free wild-type ( A ) and H72A mutant ( B ) structures. BC loop and FG helices are colored magenta and green in ligand-free and complex structures, respectively. The filipin I molecule is shown as yellow sticks . In the ligand-free wild-type structure ( A ), the side chain of His-72 is ligated to the heme iron as the sixth ligand, and the BC loop sinks into the heme to completely cover the distal pocket.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Closing motion of CYP105P1. Shown is a stereographic superimposition of the filipin I complex structure with the ligand-free wild-type ( A ) and H72A mutant ( B ) structures. BC loop and FG helices are colored magenta and green in ligand-free and complex structures, respectively. The filipin I molecule is shown as yellow sticks . In the ligand-free wild-type structure ( A ), the side chain of His-72 is ligated to the heme iron as the sixth ligand, and the BC loop sinks into the heme to completely cover the distal pocket.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques: Mutagenesis

    Spectral changes of CYP105D6 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 420 nm ( C ). A nonlinear fitting with a quadratic equation was applied to the titration curve.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Spectral changes of CYP105D6 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 420 nm ( C ). A nonlinear fitting with a quadratic equation was applied to the titration curve.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques: Titration

    Internalization of the hMCRs in HEK293 cells. Cells were incubated with [ 125 I]-NDP- α -MSH and caveolae inhibitors, filipin (▼) and nystatin (▼) compared with control (|) at 37 °C for the indicated times. Acid-resistant and acid-sensitive binding were determined. Percentage internalization (acid-resistant binding) was expressed by measuring total binding at each time-point.

    Journal: Chemical biology & drug design

    Article Title: Cell Signaling and Trafficking of Human Melanocortin Receptors in Real Time Using Two-photon Fluorescence and Confocal Laser Microscopy: Differentiation of Agonists and Antagonists

    doi: 10.1111/j.1747-0285.2006.00432.x

    Figure Lengend Snippet: Internalization of the hMCRs in HEK293 cells. Cells were incubated with [ 125 I]-NDP- α -MSH and caveolae inhibitors, filipin (▼) and nystatin (▼) compared with control (|) at 37 °C for the indicated times. Acid-resistant and acid-sensitive binding were determined. Percentage internalization (acid-resistant binding) was expressed by measuring total binding at each time-point.

    Article Snippet: H-89 and Filipin were from Calbiochem (San Diego, CA, USA).

    Techniques: Incubation, Binding Assay