Structured Review

Thermo Fisher filipin
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 <t>filipin</t> (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
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Images

1) 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

2) Product Images from "Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes. Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes"

Article Title: Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes. Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes

Journal: Physiological Reports

doi: 10.14814/phy2.13500

(A) Representative images of control (top) and M β CD ‐treated (bottom) cells stained with filipin. Scalebars represent 25 μ m; (B) Corresponding power spectra. (C) Amplitude of the first harmonic of the filipin power spectra in control and M β CD ‐treated cells ( n = 16/3 and n = 14/3, respectively). (D) Representative confocal images of control (top) and M β CD ‐treated (bottom) myocytes stained with di‐8‐ ANEPPS . Scalebars represent 20 μ m. (E) Corresponding power spectra. (F) Mean frequency and (G) amplitude of the first harmonic of the di‐8‐ ANEPPS power spectra. (H) Mean t‐tubule density. Control n = 14/3, M β CD n = 27/5, **** P
Figure Legend Snippet: (A) Representative images of control (top) and M β CD ‐treated (bottom) cells stained with filipin. Scalebars represent 25 μ m; (B) Corresponding power spectra. (C) Amplitude of the first harmonic of the filipin power spectra in control and M β CD ‐treated cells ( n = 16/3 and n = 14/3, respectively). (D) Representative confocal images of control (top) and M β CD ‐treated (bottom) myocytes stained with di‐8‐ ANEPPS . Scalebars represent 20 μ m. (E) Corresponding power spectra. (F) Mean frequency and (G) amplitude of the first harmonic of the di‐8‐ ANEPPS power spectra. (H) Mean t‐tubule density. Control n = 14/3, M β CD n = 27/5, **** P

Techniques Used: Staining

3) Product Images from "Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane ▿"

Article Title: Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00308-07

Effect of cholesterol-binding drugs on HIV-1 Gag trafficking in HEK 293T cells. HEK 293T cells were transfected with the HxBc2 provirus. Two days after transfection, cells were pretreated with filipin (4 μg/ml) or MβCD (8 mM) for 30 min
Figure Legend Snippet: Effect of cholesterol-binding drugs on HIV-1 Gag trafficking in HEK 293T cells. HEK 293T cells were transfected with the HxBc2 provirus. Two days after transfection, cells were pretreated with filipin (4 μg/ml) or MβCD (8 mM) for 30 min

Techniques Used: Binding Assay, Transfection

4) Product Images from "Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae ▿"

Article Title: Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae ▿

Journal: Eukaryotic Cell

doi: 10.1128/EC.00386-07

Colocalization of FM4-64 late endosome fluorescence and internal filipin fluorescence. Wild-type (WT; BY4741), ptc1 Δ (CBY2448), and plc1 Δ (CBY2464) strains were incubated with FM4-64 in synthetic medium for 25 min at 30°C. Cells were fixed and stained with filipin, and the coincident staining of FM4-64-fluorescent endosomes (red) and filipin-stained membranes (false-colored green) was observed by fluorescence microscopy. In wild-type cells, internal filipin-stained spots overlapped with FM4-64-fluorescent late endosomes, as shown by arrows pointing to overlapping yellow spots in the merged image. Asterisks (*) indicate examples of filipin-stained spots that did not colocalize with FM4-64. The colocalizations detected did not represent fluorescence bleed-through since FM4-64 was not detected by DAPI (4′,6′-diamidino-2-phenylindole) fluorescence and filipin was not detected by Texas red fluorescence channels (data not shown). The scale bar for all panels is 10 μm.
Figure Legend Snippet: Colocalization of FM4-64 late endosome fluorescence and internal filipin fluorescence. Wild-type (WT; BY4741), ptc1 Δ (CBY2448), and plc1 Δ (CBY2464) strains were incubated with FM4-64 in synthetic medium for 25 min at 30°C. Cells were fixed and stained with filipin, and the coincident staining of FM4-64-fluorescent endosomes (red) and filipin-stained membranes (false-colored green) was observed by fluorescence microscopy. In wild-type cells, internal filipin-stained spots overlapped with FM4-64-fluorescent late endosomes, as shown by arrows pointing to overlapping yellow spots in the merged image. Asterisks (*) indicate examples of filipin-stained spots that did not colocalize with FM4-64. The colocalizations detected did not represent fluorescence bleed-through since FM4-64 was not detected by DAPI (4′,6′-diamidino-2-phenylindole) fluorescence and filipin was not detected by Texas red fluorescence channels (data not shown). The scale bar for all panels is 10 μm.

Techniques Used: Fluorescence, Incubation, Staining, Microscopy

5) Product Images from "Uptake and Persistence of Mycobacterium avium subsp. paratuberculosis in Human Monocytes"

Article Title: Uptake and Persistence of Mycobacterium avium subsp. paratuberculosis in Human Monocytes

Journal: Infection and Immunity

doi: 10.1128/IAI.00534-12

Intracellular M. avium subsp. paratuberculosis colocalizes with cholesterol. THP-1 cells infected with FITC-labeled bacteria (MOI, 50:1) for 48 h were examined using a ×40 oil-immersion (numerical aperture, 1.3) lens and confocal microscopy (model SP5; Leica, Wetzlar, Germany). Filipin, indicated in red, was excited with 405 nm light (violet), and emission was collected from 420 to 480 nm, while FITC, shown in green, was excited with 488 nm (blue) light and fluorescence was collected from 500 to 550 nm. Bar = 5 μm.
Figure Legend Snippet: Intracellular M. avium subsp. paratuberculosis colocalizes with cholesterol. THP-1 cells infected with FITC-labeled bacteria (MOI, 50:1) for 48 h were examined using a ×40 oil-immersion (numerical aperture, 1.3) lens and confocal microscopy (model SP5; Leica, Wetzlar, Germany). Filipin, indicated in red, was excited with 405 nm light (violet), and emission was collected from 420 to 480 nm, while FITC, shown in green, was excited with 488 nm (blue) light and fluorescence was collected from 500 to 550 nm. Bar = 5 μm.

Techniques Used: Infection, Labeling, Confocal Microscopy, Fluorescence

Cholesterol aggregates at the site of M. avium subsp. paratuberculosis internalization. THP-1 cells infected with bacteria (MOI, 50:1) for 4 h were fixed, stained, and visualized for filipin-labeled cholesterol, FITC-labeled bacteria, and Texas Red-conjugated phalloidin-stained actin. The right-hand column shows an overlay image. The images, which are representative of three independent experiments, denote cells with no bacteria (A), M. bovis (B), M. avium subsp. paratuberculosis (C), and E. coli BL21 (D). Bar = 10 μm.
Figure Legend Snippet: Cholesterol aggregates at the site of M. avium subsp. paratuberculosis internalization. THP-1 cells infected with bacteria (MOI, 50:1) for 4 h were fixed, stained, and visualized for filipin-labeled cholesterol, FITC-labeled bacteria, and Texas Red-conjugated phalloidin-stained actin. The right-hand column shows an overlay image. The images, which are representative of three independent experiments, denote cells with no bacteria (A), M. bovis (B), M. avium subsp. paratuberculosis (C), and E. coli BL21 (D). Bar = 10 μm.

Techniques Used: Infection, Staining, Labeling

6) Product Images from "Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide"

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide

Journal: Autophagy

doi: 10.1080/15548627.2015.1017179

B cells internalize the P140 peptide by clathrin-mediated endocytosis. B cells from MRL/lpr mice were pretreated 30 min at 37°C with the following endocytosis inhibitors: chlorpromazine (CPZ) for clathrin-mediated endocytosis ( A ), filipin for caveolin-mediated endocytosis ( B ), and methyl-β-cyclodextrin for macropinocytosis ( C ), followed by the addition of Alexa Fluor 488-labeled P140 peptide for 30 min at 37°C. TF, bodipy, and dextran were used as respective markers of each pathway. As a control, cells were incubated at 4°C to inhibit endocytosis. For macropinocytosis analysis, unpurified splenocytes were also used since dextran was not detected into B cells. Representative results of 2 different experiments are shown.
Figure Legend Snippet: B cells internalize the P140 peptide by clathrin-mediated endocytosis. B cells from MRL/lpr mice were pretreated 30 min at 37°C with the following endocytosis inhibitors: chlorpromazine (CPZ) for clathrin-mediated endocytosis ( A ), filipin for caveolin-mediated endocytosis ( B ), and methyl-β-cyclodextrin for macropinocytosis ( C ), followed by the addition of Alexa Fluor 488-labeled P140 peptide for 30 min at 37°C. TF, bodipy, and dextran were used as respective markers of each pathway. As a control, cells were incubated at 4°C to inhibit endocytosis. For macropinocytosis analysis, unpurified splenocytes were also used since dextran was not detected into B cells. Representative results of 2 different experiments are shown.

Techniques Used: Mouse Assay, Labeling, Incubation

7) Product Images from "Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5"

Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

Journal: Virology Journal

doi: 10.1186/1743-422X-11-40

CavME endocytosis and macropinocytosis are not required for ABLV G-mediated viral entry. (A) Chemical inhibition of CavME. HEK293T cell monolayers were pretreated with filipin diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection. (B) Cholera toxin B (CTX-B) subunit uptake is inhibited by filipin. Following pretreatment with filipin as described in (A) , HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 488-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI, (4′,6-diamidino-2-phenylindole, dihydrochloride). (C) Chemical inhibition of macropinocytosis. HEK293T cell monolayers were pretreated with EIPA diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . rVSV encoding VSV G (MOI = 1) or EboGP (MOI = 15) were included as negative and positive controls, respectively, to assess EIPA activity. Drug was maintained for the entire course of infection. For (A) and (C) results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are SEM. Under these experimental conditions, the chosen MOIs yielded 60-70% virus-infected cells in untreated controls. EIPA, 5-(N-ethyl-N-isopropyl) amiloride.
Figure Legend Snippet: CavME endocytosis and macropinocytosis are not required for ABLV G-mediated viral entry. (A) Chemical inhibition of CavME. HEK293T cell monolayers were pretreated with filipin diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection. (B) Cholera toxin B (CTX-B) subunit uptake is inhibited by filipin. Following pretreatment with filipin as described in (A) , HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 488-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI, (4′,6-diamidino-2-phenylindole, dihydrochloride). (C) Chemical inhibition of macropinocytosis. HEK293T cell monolayers were pretreated with EIPA diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . rVSV encoding VSV G (MOI = 1) or EboGP (MOI = 15) were included as negative and positive controls, respectively, to assess EIPA activity. Drug was maintained for the entire course of infection. For (A) and (C) results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are SEM. Under these experimental conditions, the chosen MOIs yielded 60-70% virus-infected cells in untreated controls. EIPA, 5-(N-ethyl-N-isopropyl) amiloride.

Techniques Used: Inhibition, Infection, Incubation, Labeling, Confocal Microscopy, Staining, Activity Assay

8) Product Images from "Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration"

Article Title: Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068088

Patterns of Localization of CFH, Fib3 and cholesterol in AMD eyes vary with CFH genotype. Ph: photoreceptors; RPE: retinal pigment epithelium; BM: Bruch’s membrane. Scale bars: 50µM. A: Section of retina from (H/H) AMD donor #57985 labeled for CFH (red), Fib3 (rabbit polyclonal) (green), unesterified cholesterol (filipin, blue) and DAPI (white). Large soft drusen contain globules co-labeled for CFH and Fib3 embedded in cholesterol rich material. B: Section of retina from (H/H) AMD donor #68536 labeled as in A. This large druse also shows colocalization of CFH and Fib3 in a filipin positive region. Note that the relative intensities of CFH/Fib3 vary among deposits, leading to a range of merged colors. C: A region of macula from (Y/Y) donor #68280 with advanced wet AMD and fibrosis. There is a basal linear deposit strongly positive for Fib3 but lacking the globular structure and colocalization with CFH seen in H/H eyes. D: Section of eye from H/Y donor #68574 (wet AMD). No colocalization of CFH and Fib3. A small druse labeled with filipin (blue) is present (arrow). E,F : Foveal sections from H/Y “normal” donor #68259. E: Shows cones labeled with PNA (blue), rods labeled for rhodopsin (green), RPE labeled for RPE65 (red) and nuclei labeled with DAPI (white). Photoreceptors appear to be intact but overlie a soft druse (arrow). F : shows a region within the foveal druse showing punctate labeling for CFH (red) and Fib3 (green) in small adjacent deposits along Bruch’s membrane (arrow). Autofluorescence from lipofuscin granules in RPE is shown in magenta.
Figure Legend Snippet: Patterns of Localization of CFH, Fib3 and cholesterol in AMD eyes vary with CFH genotype. Ph: photoreceptors; RPE: retinal pigment epithelium; BM: Bruch’s membrane. Scale bars: 50µM. A: Section of retina from (H/H) AMD donor #57985 labeled for CFH (red), Fib3 (rabbit polyclonal) (green), unesterified cholesterol (filipin, blue) and DAPI (white). Large soft drusen contain globules co-labeled for CFH and Fib3 embedded in cholesterol rich material. B: Section of retina from (H/H) AMD donor #68536 labeled as in A. This large druse also shows colocalization of CFH and Fib3 in a filipin positive region. Note that the relative intensities of CFH/Fib3 vary among deposits, leading to a range of merged colors. C: A region of macula from (Y/Y) donor #68280 with advanced wet AMD and fibrosis. There is a basal linear deposit strongly positive for Fib3 but lacking the globular structure and colocalization with CFH seen in H/H eyes. D: Section of eye from H/Y donor #68574 (wet AMD). No colocalization of CFH and Fib3. A small druse labeled with filipin (blue) is present (arrow). E,F : Foveal sections from H/Y “normal” donor #68259. E: Shows cones labeled with PNA (blue), rods labeled for rhodopsin (green), RPE labeled for RPE65 (red) and nuclei labeled with DAPI (white). Photoreceptors appear to be intact but overlie a soft druse (arrow). F : shows a region within the foveal druse showing punctate labeling for CFH (red) and Fib3 (green) in small adjacent deposits along Bruch’s membrane (arrow). Autofluorescence from lipofuscin granules in RPE is shown in magenta.

Techniques Used: Labeling

9) Product Images from "Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway"

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.00903-12

Filipin and MβCD inhibit JEV entry into B104 cells. (A and C) B104 cells were pretreated with filipin (A) or MβCD (C) and infected with JEV. After 1 h of internalization, extracellular virus was inactivated with proteinase K and the cell
Figure Legend Snippet: Filipin and MβCD inhibit JEV entry into B104 cells. (A and C) B104 cells were pretreated with filipin (A) or MβCD (C) and infected with JEV. After 1 h of internalization, extracellular virus was inactivated with proteinase K and the cell

Techniques Used: Infection

10) Product Images from "Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma"

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

Journal: Cancer Cell International

doi: 10.1186/1475-2867-11-27

Effect of Filipin on uptake of TM601, CholeraToxin and EGF by glioma cells . Images of live U373 glioma cells treated with 1 μg/ml CholeraToxin B-555 (A, D), or 1 μg/ml EGF-TX-Red (B, E), or immunocytochemistry of U373 glioma treated with 10 μM TM601 (C, F) in presence or absence of filipin (5 μg/ml). There was no visible effect of filipin treatment on the level or pattern of staining for TM601, but EGF and cholera toxin staining was affected. Data are representative of total n = 4 - 5 experiments. Scale bars = 10 μm, magnification 1890 ×.
Figure Legend Snippet: Effect of Filipin on uptake of TM601, CholeraToxin and EGF by glioma cells . Images of live U373 glioma cells treated with 1 μg/ml CholeraToxin B-555 (A, D), or 1 μg/ml EGF-TX-Red (B, E), or immunocytochemistry of U373 glioma treated with 10 μM TM601 (C, F) in presence or absence of filipin (5 μg/ml). There was no visible effect of filipin treatment on the level or pattern of staining for TM601, but EGF and cholera toxin staining was affected. Data are representative of total n = 4 - 5 experiments. Scale bars = 10 μm, magnification 1890 ×.

Techniques Used: Immunocytochemistry, Staining

11) 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

Filipin-treated A549 cells have significantly fewer viable intracellular bacteria after uptake. Filipin- and non-Filipin treated epithelial cells were infected with CDC1551, HN878 and H37Rv at MOI 100 as described. Intracellular bacterial viability over time (amikacin protection assays) was quantified by lysing host cells and plating on Middlebrook 7H10 medium at T0, after 6 hr of bacterial uptake, and 24 hpi (T24)(A). Numbers of viable bacteria were significantly decreased in Filipin-treated compared to non-treated host cells infected with HN878 and H37Rv at T0 (**p-value
Figure Legend Snippet: Filipin-treated A549 cells have significantly fewer viable intracellular bacteria after uptake. Filipin- and non-Filipin treated epithelial cells were infected with CDC1551, HN878 and H37Rv at MOI 100 as described. Intracellular bacterial viability over time (amikacin protection assays) was quantified by lysing host cells and plating on Middlebrook 7H10 medium at T0, after 6 hr of bacterial uptake, and 24 hpi (T24)(A). Numbers of viable bacteria were significantly decreased in Filipin-treated compared to non-treated host cells infected with HN878 and H37Rv at T0 (**p-value

Techniques Used: Infection

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

12) Product Images from "Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells"

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026289

Hair cell cholesterol content is depleted by MβCD. (A) Representative images of filipin labeling (blue) show markedly reduced staining intensity in MβCD-treated cells. (B) Quantitative analysis shows a significant reduction in the average pixel intensity of the soma (region assessed depicted in B'). (C) Cholesterol staining peaks in the apical and basolateral ends of the cell, and MβCD reduces staining intensity by ∼50–60% while maintaining this distribution (line profile assessed depicted in B''). (D) MβCD-treatment showed an apparent loss of structural integrity at the bundle insertion at the cuticular plate (arrow highlights bundle orientation in top panels). The majority of MβCD-treated cells, as well as all untreated cells, had the expected perpendicular arrangement (bottom panels). *** = p
Figure Legend Snippet: Hair cell cholesterol content is depleted by MβCD. (A) Representative images of filipin labeling (blue) show markedly reduced staining intensity in MβCD-treated cells. (B) Quantitative analysis shows a significant reduction in the average pixel intensity of the soma (region assessed depicted in B'). (C) Cholesterol staining peaks in the apical and basolateral ends of the cell, and MβCD reduces staining intensity by ∼50–60% while maintaining this distribution (line profile assessed depicted in B''). (D) MβCD-treatment showed an apparent loss of structural integrity at the bundle insertion at the cuticular plate (arrow highlights bundle orientation in top panels). The majority of MβCD-treated cells, as well as all untreated cells, had the expected perpendicular arrangement (bottom panels). *** = p

Techniques Used: Labeling, Staining

Related Articles

Immunocytochemistry:

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma
Article Snippet: The next day, cells were pre-treated with filipin complex from Streptomyces filipinesis (Sigma-Aldrich, St. Louis MO) at 1 μg/ml for 1 h at 37°C in 5% CO2 [ ] or at 1-5 μg/ml for 15 min at 4°C [ ] followed by treatment with 10 μM TM601 (for immunocytochemistry) or 10 μM TM601-488 (for direct fluorescence) for 1 h or 24 h at 37°C in 5% CO2 . .. In addition, the effect of filipin on endocytosis of the epidermal growth factor (EGF)- biotynylated complex to Texas-Red streptavidin (TX-Red) at 1 or 10 μg/ml or CholeraToxin subunit B-555 at 1 μg/ml (both from Invitrogen, Carlsbad, CA) were evaluated.

Blocking Assay:

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: .. The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA). .. Z-stack immunofluorescence imaging was done on 20 cells in each sample.

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: Ten μg/mL CPZ (Sigma-Aldrich, C-8138) was used to inhibit clathrin-mediated endocytosis, 5 μg/mL filipin (Sigma-Aldrich, F-4767) to disrupt caveolae-dependent endocytosis, and 5 mM methyl-β-cyclodextrin (Sigma-Aldrich, C-4555) to block macropinocytosis. .. The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin.

Incubation:

Article Title: Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane ▿
Article Snippet: .. Cells were treated for 30 min with chlorpromazine (10 μg/ml), filipin (4 μg/ml), or MβCD (8 mM) at 37°C (drugs maintained throughout the experiment), subsequently washed with PBS, and incubated for 30 min at 0°C with either Tfr- or cholera toxin β subunit (ChTxβ)-Alexa-488 conjugates (Molecular Probes, Burlington, Ontario, Canada) in PBS. .. Cells were then washed with cold PBS and then incubated at 37°C for 10 min to allow Tfr or ChTxβ internalization.

Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5
Article Snippet: .. Cholera toxin B subunit uptake HEK293T cells grown on 12 mm coverslips in 24-well tissue culture plates were pretreated with filipin or transfected with Eps15 plasmids as described above and then incubated with Alexa Fluor (AF) 488- or AF 594-conjugated cholera toxin B subunit (Invitrogen) (10 μg/ml) diluted in OptiMEM® for 1 hr at 37°C. ..

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells
Article Snippet: .. Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes). .. Images were obtained with a Leica DM LB fluorescence microscope with a cooled-CCD colour digital camera (MicroPublisher, Q Imaging) with the UV filter set.

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma
Article Snippet: Filipin U373 glioma and normal NHDF cells were seeded in either the LabTekII CC2 8-glass chamber slides at 20 × 103 cells/chamber (for immunocytochemistry) or in MatTek's glass bottom dishes at 20 × 103 cells/per insert (for direct fluorescence) and incubated overnight at 37°C in 5% CO2 in growth medium containing 10% FBS. .. In addition, the effect of filipin on endocytosis of the epidermal growth factor (EGF)- biotynylated complex to Texas-Red streptavidin (TX-Red) at 1 or 10 μg/ml or CholeraToxin subunit B-555 at 1 μg/ml (both from Invitrogen, Carlsbad, CA) were evaluated.

Article Title: Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion
Article Snippet: .. After 1 hour incubation with Filipin III in the dark at room temperature (RT), the cells were washed in TBS-T, followed by detachment and mounting of the coverslips in the aqueous-based mounting medium (Clearmount™, Invitrogen, Carlsbad, CA). .. Images were taken under an Olympus Provis fluorescence microscope (Olympus Optical, Tokyo, Japan) equipped with a 40X oil objective lens.

Article Title: Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration
Article Snippet: Sections were incubated with 1 ml of filipin for 2 hours at RT in the dark and washed 3X with PBS. .. When using filipin, TO-PRO-3 (T3605 642/662 Invitrogen, 1∶500 dilutions) was as a nuclear stain.

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin. .. B104 cells were incubated with the inhibitor for 1 h at 37°C, after which the marker was added.

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin. .. For in vitro colocalization studies, B cells were incubated in the presence of 10 μM Alexa Fluor 488-labeled P140 peptide or Alexa Fluor 488-labeled peptide 131–151, for 15 min (for colocalization with EEA1) or 2 h (for colocalization with RAB9 or LAMP2/2A) at 37°C in PBS-2% FCS.

Article Title: The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis
Article Snippet: .. After 6 hr incubation with or without Filipin, 100 µg of 10,000 MW dextran-Texas Red (Invitrogen) was added to each well. ..

Infection:

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: Paragraph title: Virus infection ... The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA).

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: After the treatment, cells were infected with JEV (MOI, 0.1) for 1 h at 37°C and then washed with PBS, treated with proteinase K, and processed as described above. .. Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin.

Article Title: The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis
Article Snippet: Paragraph title: Epithelial Infection ... After 6 hr incubation with or without Filipin, 100 µg of 10,000 MW dextran-Texas Red (Invitrogen) was added to each well.

Transfection:

Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5
Article Snippet: .. Cholera toxin B subunit uptake HEK293T cells grown on 12 mm coverslips in 24-well tissue culture plates were pretreated with filipin or transfected with Eps15 plasmids as described above and then incubated with Alexa Fluor (AF) 488- or AF 594-conjugated cholera toxin B subunit (Invitrogen) (10 μg/ml) diluted in OptiMEM® for 1 hr at 37°C. ..

Amplex Red Cholesterol Assay:

Article Title: Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells
Article Snippet: .. To determine whether treatment of TNBC cells with different concentrations of MβCD, nystatin, and filipin III efficiently extracted cellular cholesterol, and to asses residual cholesterol levels 48 hours later, we assayed cellular cholesterol levels using an Amplex® Red Cholesterol Assay kit (Invitrogen). .. As shown in , extraction of cellular cholesterol increased with increasing MβCD concentration in a dose-dependent manner at 1, 24, and 48-hours in both cell lines.

Cell Culture:

Article Title: Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae ▿
Article Snippet: .. For filipin and FM4-64 colocalization, 5.0 units of log-phase cells at an optical density at 600 nm grown in synthetic complete medium at 30°C were pelleted and cultured at 30°C with 32 μM FXM4-64 (Molecular Probes/Invitrogen, Carlsbad, CA) ( ) for either 5 or 25 min. After the timed FXM4-64 uptake, cells were washed once with water, pelleted, and diluted to an optical density at 600 nm of 0.7 units/ml with fresh medium. ..

Generated:

Article Title: Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development
Article Snippet: Staining of liver tissue sections Liver tissue sections were generated from U18666A-treated and control animals. .. Briefly, after fixing for 15 min in Formalin, sections were washed with PBS, and 0.05 mg/ml filipin was added to the sections for 1 h. Slides were mounted using ProLong Gold Antifade Mountant (Life Technologies) and examined using an Olympus BX50 microscope.

Inhibition:

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: For endocytosis inhibition studies, total splenocytes or purified B cells were pretreated with endocytosis inhibitors for 30 min at 37°C, and then loaded with Alexa Fluor 488-labeled P140 peptide at 10 μM for 30 min at 37°C. .. The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin.

Imaging:

Article Title: Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes. Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes
Article Snippet: Cells were fixed using 4% paraformaldehyde for 10 min before incubating in 0.05 mg/mL filipin in 10% fetal bovine serum for 2 h. Cells were mounted with Prolong Gold before imaging on a Leica SP8 tandem scanning system with a 1.1 numerical aperture 40 × water immersion objective. .. Filipin was excited with a Spectra Physics DeepSee tuneable multiphoton laser set to 760 nm and light collected between 451 and 515 nm.

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells
Article Snippet: Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes). .. Images were obtained with a Leica DM LB fluorescence microscope with a cooled-CCD colour digital camera (MicroPublisher, Q Imaging) with the UV filter set.

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA). .. Z-stack immunofluorescence imaging was done on 20 cells in each sample.

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin. .. The incubation was continued for 30 min, the cells were fixed and stained with DAPI (4′,6-diamidino-2-phenylindole) (Roche), and the data were analyzed by confocal imaging.

Immunofluorescence:

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA). .. Z-stack immunofluorescence imaging was done on 20 cells in each sample.

Article Title: Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration
Article Snippet: Paragraph title: Immunofluorescence ... When using filipin, TO-PRO-3 (T3605 642/662 Invitrogen, 1∶500 dilutions) was as a nuclear stain.

Molecular Weight:

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma
Article Snippet: In addition, the effect of filipin on endocytosis of the epidermal growth factor (EGF)- biotynylated complex to Texas-Red streptavidin (TX-Red) at 1 or 10 μg/ml or CholeraToxin subunit B-555 at 1 μg/ml (both from Invitrogen, Carlsbad, CA) were evaluated. .. EGF was selected with a similar molecular weight (~6000 kDa) as TM601 (~4000 kDa).

Fluorescence:

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells
Article Snippet: Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes). .. Images were obtained with a Leica DM LB fluorescence microscope with a cooled-CCD colour digital camera (MicroPublisher, Q Imaging) with the UV filter set.

Article Title: Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae ▿
Article Snippet: Paragraph title: Filipin/sterol and Nile red fluorescence microscopy. ... For filipin and FM4-64 colocalization, 5.0 units of log-phase cells at an optical density at 600 nm grown in synthetic complete medium at 30°C were pelleted and cultured at 30°C with 32 μM FXM4-64 (Molecular Probes/Invitrogen, Carlsbad, CA) ( ) for either 5 or 25 min. After the timed FXM4-64 uptake, cells were washed once with water, pelleted, and diluted to an optical density at 600 nm of 0.7 units/ml with fresh medium.

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma
Article Snippet: The next day, cells were pre-treated with filipin complex from Streptomyces filipinesis (Sigma-Aldrich, St. Louis MO) at 1 μg/ml for 1 h at 37°C in 5% CO2 [ ] or at 1-5 μg/ml for 15 min at 4°C [ ] followed by treatment with 10 μM TM601 (for immunocytochemistry) or 10 μM TM601-488 (for direct fluorescence) for 1 h or 24 h at 37°C in 5% CO2 . .. In addition, the effect of filipin on endocytosis of the epidermal growth factor (EGF)- biotynylated complex to Texas-Red streptavidin (TX-Red) at 1 or 10 μg/ml or CholeraToxin subunit B-555 at 1 μg/ml (both from Invitrogen, Carlsbad, CA) were evaluated.

Article Title: Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion
Article Snippet: After 1 hour incubation with Filipin III in the dark at room temperature (RT), the cells were washed in TBS-T, followed by detachment and mounting of the coverslips in the aqueous-based mounting medium (Clearmount™, Invitrogen, Carlsbad, CA). .. Images were taken under an Olympus Provis fluorescence microscope (Olympus Optical, Tokyo, Japan) equipped with a 40X oil objective lens.

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: The cells then were washed twice with PBS, mounted on PBS, and visualized on a Zeiss LSM-710 fluorescence microscope. .. Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin.

Multiple Displacement Amplification:

Article Title: Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells
Article Snippet: Effect of lipid raft disrupting agents on cellular cholesterol levels We estimated the levels of cholesterol in normal (MCF 12A) and TNBC cell lines (MDA-MB 231 & MDA-MB 468), we found that TNBC cell lines exhibited higher ratios of cholesterol than the normal cell line ( ). .. To determine whether treatment of TNBC cells with different concentrations of MβCD, nystatin, and filipin III efficiently extracted cellular cholesterol, and to asses residual cholesterol levels 48 hours later, we assayed cellular cholesterol levels using an Amplex® Red Cholesterol Assay kit (Invitrogen).

CtB Assay:

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: .. Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin. .. B104 cells were incubated with the inhibitor for 1 h at 37°C, after which the marker was added.

Detection Assay:

Article Title: Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion
Article Snippet: After 24-hours incubation, the cells were stained using the cholesterol cell-based detection assay kit (Cayman Chemical) according to the manufacturer's instructions. .. After 1 hour incubation with Filipin III in the dark at room temperature (RT), the cells were washed in TBS-T, followed by detachment and mounting of the coverslips in the aqueous-based mounting medium (Clearmount™, Invitrogen, Carlsbad, CA).

Microscopy:

Article Title: Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane ▿
Article Snippet: Cells were treated for 30 min with chlorpromazine (10 μg/ml), filipin (4 μg/ml), or MβCD (8 mM) at 37°C (drugs maintained throughout the experiment), subsequently washed with PBS, and incubated for 30 min at 0°C with either Tfr- or cholera toxin β subunit (ChTxβ)-Alexa-488 conjugates (Molecular Probes, Burlington, Ontario, Canada) in PBS. .. Cells were examined by conventional epifluorescence micrographs on a Zeiss Cell Observer system (Zeiss, Toronto, Ontario, Canada) equipped with an Axiovert 200 M microscope and a Zeiss Axiocam ultra-high-resolution monochrome digital camera, using the ×100 oil lens.

Article Title: Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes. Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes
Article Snippet: Filipin was excited with a Spectra Physics DeepSee tuneable multiphoton laser set to 760 nm and light collected between 451 and 515 nm. .. Cells were imaged on an LSM 880 confocal microscope (Zeiss, Germany) in Airyscan “super‐resolution” mode, with a 1.3 numerical aperture 63 × oil immersion objective, with voxel size set to 40 nm in‐plane and 180 nm along the optical axis.

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells
Article Snippet: Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes). .. Images were obtained with a Leica DM LB fluorescence microscope with a cooled-CCD colour digital camera (MicroPublisher, Q Imaging) with the UV filter set.

Article Title: Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae ▿
Article Snippet: Paragraph title: Filipin/sterol and Nile red fluorescence microscopy. ... For filipin and FM4-64 colocalization, 5.0 units of log-phase cells at an optical density at 600 nm grown in synthetic complete medium at 30°C were pelleted and cultured at 30°C with 32 μM FXM4-64 (Molecular Probes/Invitrogen, Carlsbad, CA) ( ) for either 5 or 25 min. After the timed FXM4-64 uptake, cells were washed once with water, pelleted, and diluted to an optical density at 600 nm of 0.7 units/ml with fresh medium.

Article Title: Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion
Article Snippet: After 1 hour incubation with Filipin III in the dark at room temperature (RT), the cells were washed in TBS-T, followed by detachment and mounting of the coverslips in the aqueous-based mounting medium (Clearmount™, Invitrogen, Carlsbad, CA). .. Images were taken under an Olympus Provis fluorescence microscope (Olympus Optical, Tokyo, Japan) equipped with a 40X oil objective lens.

Article Title: Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development
Article Snippet: .. Briefly, after fixing for 15 min in Formalin, sections were washed with PBS, and 0.05 mg/ml filipin was added to the sections for 1 h. Slides were mounted using ProLong Gold Antifade Mountant (Life Technologies) and examined using an Olympus BX50 microscope. ..

Article Title: Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration
Article Snippet: When using filipin, TO-PRO-3 (T3605 642/662 Invitrogen, 1∶500 dilutions) was as a nuclear stain. .. They were stained with Gill’s haematoxylin (Vector Labs) for 5 minutes, rinsed in water, dipped three times in ammonia solution, rinsed and stained with Eosin Y (Baker Analyzed) for 3 minutes, dehydrated in 5 quick dips in 2 changes of 95% ethanol and 2 changes of 100% ethanol then 10 dips in 2 changes of xylene, cleared and mounted using VectaMount H-5000 (Vector Labs) and imaged using a BX51 Olympus microscope.

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: The cells then were washed twice with PBS, mounted on PBS, and visualized on a Zeiss LSM-710 fluorescence microscope. .. Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin.

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin. .. Images were acquired with a Zeiss Axiovert LSM700 confocal microscope (Jena, Germany) with a 63× oil immersion objective.

Purification:

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: For endocytosis inhibition studies, total splenocytes or purified B cells were pretreated with endocytosis inhibitors for 30 min at 37°C, and then loaded with Alexa Fluor 488-labeled P140 peptide at 10 μM for 30 min at 37°C. .. The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin.

Confocal Microscopy:

Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5
Article Snippet: Cholera toxin B subunit uptake HEK293T cells grown on 12 mm coverslips in 24-well tissue culture plates were pretreated with filipin or transfected with Eps15 plasmids as described above and then incubated with Alexa Fluor (AF) 488- or AF 594-conjugated cholera toxin B subunit (Invitrogen) (10 μg/ml) diluted in OptiMEM® for 1 hr at 37°C. .. The coverslips were mounted on slides using SouthernBiotech™ Dapi-Fluoromount-G™ clear mounting media and imaged by confocal microscopy.

Mouse Assay:

Article Title: Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development
Article Snippet: At 42 h postinfection, mice were killed and perfused with PBS. .. Briefly, after fixing for 15 min in Formalin, sections were washed with PBS, and 0.05 mg/ml filipin was added to the sections for 1 h. Slides were mounted using ProLong Gold Antifade Mountant (Life Technologies) and examined using an Olympus BX50 microscope.

Plasmid Preparation:

Article Title: Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration
Article Snippet: When using filipin, TO-PRO-3 (T3605 642/662 Invitrogen, 1∶500 dilutions) was as a nuclear stain. .. They were stained with Gill’s haematoxylin (Vector Labs) for 5 minutes, rinsed in water, dipped three times in ammonia solution, rinsed and stained with Eosin Y (Baker Analyzed) for 3 minutes, dehydrated in 5 quick dips in 2 changes of 95% ethanol and 2 changes of 100% ethanol then 10 dips in 2 changes of xylene, cleared and mounted using VectaMount H-5000 (Vector Labs) and imaged using a BX51 Olympus microscope.

Software:

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells
Article Snippet: Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes). .. Staining intensity was measured with ImageJ software, where the average pixel intensity of the soma and the line profile intensity as a function of distance from the basolateral pole to the bottom edge of the cuticular plate were measured.

Article Title: Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion
Article Snippet: After 1 hour incubation with Filipin III in the dark at room temperature (RT), the cells were washed in TBS-T, followed by detachment and mounting of the coverslips in the aqueous-based mounting medium (Clearmount™, Invitrogen, Carlsbad, CA). .. Quantification of the cholesterol levels was accomplished using the Granularity algorithm in the MetaXpress image analysis software (Molecular Devices Corp., Sunnyvale CA).

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA). .. 3D image reconstruction was performed using Imaris software (Bitplane, Zurich, Switzerland) after image deconvolution by AutoQuant X3 software (MediaCybernetics, Rockville, MD).

Irradiation:

Article Title: The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis
Article Snippet: After 6 hr incubation with or without Filipin, 100 µg of 10,000 MW dextran-Texas Red (Invitrogen) was added to each well. .. For consistency, cold synchronization of A549 cells was employed prior to the addition of live or gamma irradiated bacteria and culture filtrate protein (CFP) or total lipid (TL) reagents (see below).

In Vitro:

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin. .. For in vitro colocalization studies, B cells were incubated in the presence of 10 μM Alexa Fluor 488-labeled P140 peptide or Alexa Fluor 488-labeled peptide 131–151, for 15 min (for colocalization with EEA1) or 2 h (for colocalization with RAB9 or LAMP2/2A) at 37°C in PBS-2% FCS.

Produced:

Article Title: The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis
Article Snippet: Studies showed no deleterious effects produced by the drug at the dose utilized (data not shown). .. After 6 hr incubation with or without Filipin, 100 µg of 10,000 MW dextran-Texas Red (Invitrogen) was added to each well.

Concentration Assay:

Article Title: Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells
Article Snippet: To determine whether treatment of TNBC cells with different concentrations of MβCD, nystatin, and filipin III efficiently extracted cellular cholesterol, and to asses residual cholesterol levels 48 hours later, we assayed cellular cholesterol levels using an Amplex® Red Cholesterol Assay kit (Invitrogen). .. As shown in , extraction of cellular cholesterol increased with increasing MβCD concentration in a dose-dependent manner at 1, 24, and 48-hours in both cell lines.

Article Title: Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae ▿
Article Snippet: For filipin and FM4-64 colocalization, 5.0 units of log-phase cells at an optical density at 600 nm grown in synthetic complete medium at 30°C were pelleted and cultured at 30°C with 32 μM FXM4-64 (Molecular Probes/Invitrogen, Carlsbad, CA) ( ) for either 5 or 25 min. After the timed FXM4-64 uptake, cells were washed once with water, pelleted, and diluted to an optical density at 600 nm of 0.7 units/ml with fresh medium. .. Cells from mid-logarithmic-phase-grown cultures were centrifuged, and the cell pellet was resuspended with water before the addition of Nile red to a final concentration of 2 μg/ml.

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: To test the effect of U18666A on virus infection, SW13 cells grown in 8-chamber μ-slides (ibidi, Munich, Germany) were treated in duplicate with U18666A to the final concentration of 30 µM or with H2 O (control). .. The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA).

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: In some experiments, Alexa Fluor 488-labeled ScP140 control peptide was used at the same concentration. .. The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin.

Article Title: The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis
Article Snippet: Control experiments with DMSO alone at the concentration applied in Filipin studies produced no cytotoxic effects. .. After 6 hr incubation with or without Filipin, 100 µg of 10,000 MW dextran-Texas Red (Invitrogen) was added to each well.

Protease Inhibitor:

Article Title: Uptake and Persistence of Mycobacterium avium subsp. paratuberculosis in Human Monocytes
Article Snippet: CDP-Star substrate, fluorescein isothiocyanate (FITC), LysoTracker Red, filipin, Texas Red-conjugated phalloidin, and Hoechst stain were purchased from Invitrogen (Carlsbad, CA). .. The protease inhibitor cocktail and the enzymatic colorimetric reagent for measuring cholesterol (CHOD-PAP) were from Roche (Manheim, Germany), gentamicin was from Pfizer (Bentley, WA, Australia), and simvastatin and methyl-β-cyclodextrin (MβCD) were from Sigma (St. Louis, MO).

Marker:

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin. .. B104 cells were incubated with the inhibitor for 1 h at 37°C, after which the marker was added.

Staining:

Article Title: Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane ▿
Article Snippet: Cells were treated for 30 min with chlorpromazine (10 μg/ml), filipin (4 μg/ml), or MβCD (8 mM) at 37°C (drugs maintained throughout the experiment), subsequently washed with PBS, and incubated for 30 min at 0°C with either Tfr- or cholera toxin β subunit (ChTxβ)-Alexa-488 conjugates (Molecular Probes, Burlington, Ontario, Canada) in PBS. .. Cytospin, paraformaldehyde fixation, staining of nuclei, and mounting were performed as previously described ( ).

Article Title: Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes. Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t‐tubule membranes in mouse ventricular myocytes
Article Snippet: Paragraph title: Membrane staining ... Filipin was excited with a Spectra Physics DeepSee tuneable multiphoton laser set to 760 nm and light collected between 451 and 515 nm.

Article Title: Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells
Article Snippet: Paragraph title: Filipin staining ... Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes).

Article Title: Uptake and Persistence of Mycobacterium avium subsp. paratuberculosis in Human Monocytes
Article Snippet: .. CDP-Star substrate, fluorescein isothiocyanate (FITC), LysoTracker Red, filipin, Texas Red-conjugated phalloidin, and Hoechst stain were purchased from Invitrogen (Carlsbad, CA). .. The protease inhibitor cocktail and the enzymatic colorimetric reagent for measuring cholesterol (CHOD-PAP) were from Roche (Manheim, Germany), gentamicin was from Pfizer (Bentley, WA, Australia), and simvastatin and methyl-β-cyclodextrin (MβCD) were from Sigma (St. Louis, MO).

Article Title: Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion
Article Snippet: Paragraph title: Cholesterol staining ... After 1 hour incubation with Filipin III in the dark at room temperature (RT), the cells were washed in TBS-T, followed by detachment and mounting of the coverslips in the aqueous-based mounting medium (Clearmount™, Invitrogen, Carlsbad, CA).

Article Title: Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators
Article Snippet: .. The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA). .. Z-stack immunofluorescence imaging was done on 20 cells in each sample.

Article Title: Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development
Article Snippet: Paragraph title: Staining of liver tissue sections ... Briefly, after fixing for 15 min in Formalin, sections were washed with PBS, and 0.05 mg/ml filipin was added to the sections for 1 h. Slides were mounted using ProLong Gold Antifade Mountant (Life Technologies) and examined using an Olympus BX50 microscope.

Article Title: Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration
Article Snippet: .. When using filipin, TO-PRO-3 (T3605 642/662 Invitrogen, 1∶500 dilutions) was as a nuclear stain. .. Adjacent cryosections were also used for hematoxylin and eosin (H & E) staining to illustrate histology.

Article Title: Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway
Article Snippet: To assess the effect of chloroquine and ammonium chloride on the pH of acid intracellular vesicles, B104 cells, treated or not with the compound for 1 h at 37°C, were stained with acridine orange (1 mg/ml in DMEM without serum) for 15 min at 37°C. .. Alexa Fluor 488 (AF 488)-conjugated transferrin (10 μg/ml) (Molecular Probes) was used to control the function of chlorpromazine and dynasore, AF 555-conjugated cholera toxin B (CTB) (10 μg/ml) (Molecular Probes) was used as a control for filipin and MβCD, and Alexa Fluor 555 (AF 555)-conjugated dextran (200 μg/ml; Molecular Probes) was used as a control for EIPA and wortmannin.

Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
Article Snippet: The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin. .. Cells were then permeabilized with 0.05% (v/v) of Triton X-100 (Pharmacia Biotech, 17–1315–01), stained with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, D1306), and finally mounted on glass slides with fluorescent mounting medium (DAKO, S302380).

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  • 90
    Thermo Fisher filipin
    Effect of CEP on free cholesterol and LDL trafficking in endothelial cells. (A–C) HUVEC were treated with CEP (5 μM) or DMSO (CTRL or control) for 14 h and observed under a confocal microscope after immunostaining with <t>filipin,</t> LAMP1, a late endolysosome marker (A), PDI, an endoplasmic reticulum (ER) marker (B), and GM130, a Golgi marker (C). Scale bar = 50 μm. Images in the inlets (red square) were magnified and shown on the right side of each figure. Filipin is shown as green and each organelle marker is shown as red color. The bottom images are merged color images. Scale bar for the inlets = 10 μm. (D–G) HUVEC were treated with CEP (5 μM) or DMSO for 8 h and then the cells were incubated with DiI-LDL for additional 1 h (D and E) or 6 h (F and G), as indicated with the time frame shown at the bottom of each figure. Filipin is shown as green and DiI-LDL is shown as red color. Scale bar = 50 μm.
    Filipin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher filipin staining
    5A-SM mobilizes cholesterol in vivo and ameliorates disease phenotypes . a Serum cholesterol from 7-week-old Npc1 I1061T homozygous mice pre- and 2 h post-treatment with 100 mg/kg 5A-SM, i.p. b Pre- (dashed line) and 2 h post-treatment (solid line) serum was fractionated by HPLC, and cholesterol was quantified by cholesterol oxidase assay. VLDL, LDL, and HDL fractions are indicated by arrows. c , d Seven-week-old wild-type (WT) and Npc1 I1061T homozygous (NPC) mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM. 48 h later, c liver HMGCS transcript levels and d total serum bilirubin were analyzed. e WT and Npc1 I1061T mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM three times per week from 7 to 11 weeks of age. The change in weight of each mouse from week 7 ( t = 0) to week 11 ( t = 4) was quantified. f Seven-week-old WT and NPC mice were injected with Veh or 100 mg/kg 5A-SM three times per week for 2 weeks. At 9 weeks of age livers were stained for macrophages using F4/80 (green) and DNA Hoechst (blue). Macrophage area is quantified at right. Scale bar = 50 μm. Violin plot shows median (dashed line), 25% and 75% (dotted lines), and probability density (thickness). g Brain slices from 8-week-old Npc1 I1061T mice were incubated with vehicle (Veh) or 5 mg/ml 5A-SM for 4 days, and <t>filipin</t> levels in Purkinje neuron soma were quantified (see also Additional file 1 : Figure S4b). h Six- to 7-week-old WT and Npc1 I1061T mice received intraventricular injections with vehicle (Veh) or 5A-SM-DiD. N: WT = 4, NPC Veh = 5, NPC 5A-SM = 4 mice. One-week later, cholesterol levels in Purkinje neuron soma (green) were analyzed by filipin (blue) staining. Dashed lines indicate Purkinje neuron soma (also see Additional file 1 : Figure S6a). Scale bar = 50 μm. Data quantified at right. Data are mean ± s.e.m. from a , b , c three; d genotype and treatment: number of mice, WT + Veh = 5, WT + 5A-SM = 3, NPC + Veh = 4, NPC + 5A-SM = 7; e genotype and treatment: number of mice at 9 weeks and 11 weeks, WT + Veh: 13 and 8, WT + 5A-SM: 9 and 8, NPC + Veh: 6, NPC + 5A-SM: 12 and 10 mice; f genotype and treatment: number of mice, cells, WT + Veh: 4, 301, NPC + Veh: 4, 514, NPC + 5A-SM: 3, 373 ( g ) WT = 93, NPC Veh = 143, NPC + 5A-SM = 116 cells. * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001. a , c Student’s t test ( t = ( a ) 6.375, ( c ) 5.23); d , f , g , h one-way ANOVA with Tukey post hoc test ( F , df = ( d ) 13.28, 3; ( f ) 368.1, 2 ( g ) 38.89, 2; ( h ) 108.3, 2); e two-way ANOVA with Bonferroni post hoc test ( F , df = 7.12, 2)
    Filipin Staining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin staining/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
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    82
    Thermo Fisher filipin labeling solution
    5A-SM mobilizes cholesterol in vivo and ameliorates disease phenotypes . a Serum cholesterol from 7-week-old Npc1 I1061T homozygous mice pre- and 2 h post-treatment with 100 mg/kg 5A-SM, i.p. b Pre- (dashed line) and 2 h post-treatment (solid line) serum was fractionated by HPLC, and cholesterol was quantified by cholesterol oxidase assay. VLDL, LDL, and HDL fractions are indicated by arrows. c , d Seven-week-old wild-type (WT) and Npc1 I1061T homozygous (NPC) mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM. 48 h later, c liver HMGCS transcript levels and d total serum bilirubin were analyzed. e WT and Npc1 I1061T mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM three times per week from 7 to 11 weeks of age. The change in weight of each mouse from week 7 ( t = 0) to week 11 ( t = 4) was quantified. f Seven-week-old WT and NPC mice were injected with Veh or 100 mg/kg 5A-SM three times per week for 2 weeks. At 9 weeks of age livers were stained for macrophages using F4/80 (green) and DNA Hoechst (blue). Macrophage area is quantified at right. Scale bar = 50 μm. Violin plot shows median (dashed line), 25% and 75% (dotted lines), and probability density (thickness). g Brain slices from 8-week-old Npc1 I1061T mice were incubated with vehicle (Veh) or 5 mg/ml 5A-SM for 4 days, and <t>filipin</t> levels in Purkinje neuron soma were quantified (see also Additional file 1 : Figure S4b). h Six- to 7-week-old WT and Npc1 I1061T mice received intraventricular injections with vehicle (Veh) or 5A-SM-DiD. N: WT = 4, NPC Veh = 5, NPC 5A-SM = 4 mice. One-week later, cholesterol levels in Purkinje neuron soma (green) were analyzed by filipin (blue) staining. Dashed lines indicate Purkinje neuron soma (also see Additional file 1 : Figure S6a). Scale bar = 50 μm. Data quantified at right. Data are mean ± s.e.m. from a , b , c three; d genotype and treatment: number of mice, WT + Veh = 5, WT + 5A-SM = 3, NPC + Veh = 4, NPC + 5A-SM = 7; e genotype and treatment: number of mice at 9 weeks and 11 weeks, WT + Veh: 13 and 8, WT + 5A-SM: 9 and 8, NPC + Veh: 6, NPC + 5A-SM: 12 and 10 mice; f genotype and treatment: number of mice, cells, WT + Veh: 4, 301, NPC + Veh: 4, 514, NPC + 5A-SM: 3, 373 ( g ) WT = 93, NPC Veh = 143, NPC + 5A-SM = 116 cells. * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001. a , c Student’s t test ( t = ( a ) 6.375, ( c ) 5.23); d , f , g , h one-way ANOVA with Tukey post hoc test ( F , df = ( d ) 13.28, 3; ( f ) 368.1, 2 ( g ) 38.89, 2; ( h ) 108.3, 2); e two-way ANOVA with Bonferroni post hoc test ( F , df = 7.12, 2)
    Filipin Labeling Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of CEP on free cholesterol and LDL trafficking in endothelial cells. (A–C) HUVEC were treated with CEP (5 μM) or DMSO (CTRL or control) for 14 h and observed under a confocal microscope after immunostaining with filipin, LAMP1, a late endolysosome marker (A), PDI, an endoplasmic reticulum (ER) marker (B), and GM130, a Golgi marker (C). Scale bar = 50 μm. Images in the inlets (red square) were magnified and shown on the right side of each figure. Filipin is shown as green and each organelle marker is shown as red color. The bottom images are merged color images. Scale bar for the inlets = 10 μm. (D–G) HUVEC were treated with CEP (5 μM) or DMSO for 8 h and then the cells were incubated with DiI-LDL for additional 1 h (D and E) or 6 h (F and G), as indicated with the time frame shown at the bottom of each figure. Filipin is shown as green and DiI-LDL is shown as red color. Scale bar = 50 μm.

    Journal: Cancer letters

    Article Title: Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth

    doi: 10.1016/j.canlet.2017.09.009

    Figure Lengend Snippet: Effect of CEP on free cholesterol and LDL trafficking in endothelial cells. (A–C) HUVEC were treated with CEP (5 μM) or DMSO (CTRL or control) for 14 h and observed under a confocal microscope after immunostaining with filipin, LAMP1, a late endolysosome marker (A), PDI, an endoplasmic reticulum (ER) marker (B), and GM130, a Golgi marker (C). Scale bar = 50 μm. Images in the inlets (red square) were magnified and shown on the right side of each figure. Filipin is shown as green and each organelle marker is shown as red color. The bottom images are merged color images. Scale bar for the inlets = 10 μm. (D–G) HUVEC were treated with CEP (5 μM) or DMSO for 8 h and then the cells were incubated with DiI-LDL for additional 1 h (D and E) or 6 h (F and G), as indicated with the time frame shown at the bottom of each figure. Filipin is shown as green and DiI-LDL is shown as red color. Scale bar = 50 μm.

    Article Snippet: After fixation and staining with filipin, cells were washed with PBS, mounted with Immu-mount (Thermo Fisher Scientific), and observed under the Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Thornwood, NY).

    Techniques: Microscopy, Immunostaining, Marker, Incubation

    Effect of CEP on mTOR subcellular distribution and signaling. (A) HUVEC were treated with CEP or DMSO for 24 h and then were immunostained with filipin (gray) and mTOR antibody (green). mTOR in control HUVEC is shown as punctae. Scale bar = 20 μm. (B) HUVEC were treated with CEP (5 μM) in the presence or absence of cholesterol-cyclodextrin complex (CHOL) in different time points and then immunostained with mTOR antibody (green), LAMP1 antibody (red) and filipin (gray). Scale bar = 20 μm. (C) HUVEC were treated with CEP (5 μM) in the presence or absence of cholesterol-cyclodextrin complex (CHOL) in different time points and then analyzed with Western blots for an mTOR substrate, 4EBP1. (D) HUVEC were treated with CEP (2.5 and 5 μM) in the presence or absence of cholesterol-cyclodextrin complex (CHOL) for 24 h and then analyzed with Western blots for 4EBP1, phospho-S6 kinase (pS6K) and total S6 kinase (S6K). GAPDH was used as an internal loading control.

    Journal: Cancer letters

    Article Title: Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth

    doi: 10.1016/j.canlet.2017.09.009

    Figure Lengend Snippet: Effect of CEP on mTOR subcellular distribution and signaling. (A) HUVEC were treated with CEP or DMSO for 24 h and then were immunostained with filipin (gray) and mTOR antibody (green). mTOR in control HUVEC is shown as punctae. Scale bar = 20 μm. (B) HUVEC were treated with CEP (5 μM) in the presence or absence of cholesterol-cyclodextrin complex (CHOL) in different time points and then immunostained with mTOR antibody (green), LAMP1 antibody (red) and filipin (gray). Scale bar = 20 μm. (C) HUVEC were treated with CEP (5 μM) in the presence or absence of cholesterol-cyclodextrin complex (CHOL) in different time points and then analyzed with Western blots for an mTOR substrate, 4EBP1. (D) HUVEC were treated with CEP (2.5 and 5 μM) in the presence or absence of cholesterol-cyclodextrin complex (CHOL) for 24 h and then analyzed with Western blots for 4EBP1, phospho-S6 kinase (pS6K) and total S6 kinase (S6K). GAPDH was used as an internal loading control.

    Article Snippet: After fixation and staining with filipin, cells were washed with PBS, mounted with Immu-mount (Thermo Fisher Scientific), and observed under the Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Thornwood, NY).

    Techniques: Western Blot

    Identification of cholesterol trafficking inhibitors in endothelial cells. (A) A schematic illustration of the screening procedure is shown. The John Hopkins Drug Library (JHDL) composed of 3,131 clinical drugs was used for screening to identify inhibitors of cholesterol trafficking in HUVEC. Finally, 13 hits were identified from primary and validation screenings. (B) HUVEC were treated with the hits for 8 h and observed under a confocal microscope after staining with filipin, a cholesterol tracer. CTRL, control; TFP, trifluoperazine; QAC, quinacrine; PCP, prochlorperazine; SLS, solasodine; TMT, tomatidine; ITRA, itraconazole; AST, astemizole; CEP, cepharanthine; NCS, niclosamide; STR, sertraline; SKF, zolantidine. Scale bar = 50 μm.

    Journal: Cancer letters

    Article Title: Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth

    doi: 10.1016/j.canlet.2017.09.009

    Figure Lengend Snippet: Identification of cholesterol trafficking inhibitors in endothelial cells. (A) A schematic illustration of the screening procedure is shown. The John Hopkins Drug Library (JHDL) composed of 3,131 clinical drugs was used for screening to identify inhibitors of cholesterol trafficking in HUVEC. Finally, 13 hits were identified from primary and validation screenings. (B) HUVEC were treated with the hits for 8 h and observed under a confocal microscope after staining with filipin, a cholesterol tracer. CTRL, control; TFP, trifluoperazine; QAC, quinacrine; PCP, prochlorperazine; SLS, solasodine; TMT, tomatidine; ITRA, itraconazole; AST, astemizole; CEP, cepharanthine; NCS, niclosamide; STR, sertraline; SKF, zolantidine. Scale bar = 50 μm.

    Article Snippet: After fixation and staining with filipin, cells were washed with PBS, mounted with Immu-mount (Thermo Fisher Scientific), and observed under the Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Thornwood, NY).

    Techniques: Microscopy, Staining, AST Assay

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1004390

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

    Article Snippet: The block of cholesterol transport out of the MVBs in the U18666A-treated cells was confirmed by staining the second set of cells with anti-CD63 antibody followed by an Alexa Flour-conjugated secondary antibody, filipin III (Thermo Fisher Scientific, Waltham, MA), and CellMask red dye (Life Technologies, Carlsbad, CA).

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

    5A-SM mobilizes cholesterol in vivo and ameliorates disease phenotypes . a Serum cholesterol from 7-week-old Npc1 I1061T homozygous mice pre- and 2 h post-treatment with 100 mg/kg 5A-SM, i.p. b Pre- (dashed line) and 2 h post-treatment (solid line) serum was fractionated by HPLC, and cholesterol was quantified by cholesterol oxidase assay. VLDL, LDL, and HDL fractions are indicated by arrows. c , d Seven-week-old wild-type (WT) and Npc1 I1061T homozygous (NPC) mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM. 48 h later, c liver HMGCS transcript levels and d total serum bilirubin were analyzed. e WT and Npc1 I1061T mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM three times per week from 7 to 11 weeks of age. The change in weight of each mouse from week 7 ( t = 0) to week 11 ( t = 4) was quantified. f Seven-week-old WT and NPC mice were injected with Veh or 100 mg/kg 5A-SM three times per week for 2 weeks. At 9 weeks of age livers were stained for macrophages using F4/80 (green) and DNA Hoechst (blue). Macrophage area is quantified at right. Scale bar = 50 μm. Violin plot shows median (dashed line), 25% and 75% (dotted lines), and probability density (thickness). g Brain slices from 8-week-old Npc1 I1061T mice were incubated with vehicle (Veh) or 5 mg/ml 5A-SM for 4 days, and filipin levels in Purkinje neuron soma were quantified (see also Additional file 1 : Figure S4b). h Six- to 7-week-old WT and Npc1 I1061T mice received intraventricular injections with vehicle (Veh) or 5A-SM-DiD. N: WT = 4, NPC Veh = 5, NPC 5A-SM = 4 mice. One-week later, cholesterol levels in Purkinje neuron soma (green) were analyzed by filipin (blue) staining. Dashed lines indicate Purkinje neuron soma (also see Additional file 1 : Figure S6a). Scale bar = 50 μm. Data quantified at right. Data are mean ± s.e.m. from a , b , c three; d genotype and treatment: number of mice, WT + Veh = 5, WT + 5A-SM = 3, NPC + Veh = 4, NPC + 5A-SM = 7; e genotype and treatment: number of mice at 9 weeks and 11 weeks, WT + Veh: 13 and 8, WT + 5A-SM: 9 and 8, NPC + Veh: 6, NPC + 5A-SM: 12 and 10 mice; f genotype and treatment: number of mice, cells, WT + Veh: 4, 301, NPC + Veh: 4, 514, NPC + 5A-SM: 3, 373 ( g ) WT = 93, NPC Veh = 143, NPC + 5A-SM = 116 cells. * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001. a , c Student’s t test ( t = ( a ) 6.375, ( c ) 5.23); d , f , g , h one-way ANOVA with Tukey post hoc test ( F , df = ( d ) 13.28, 3; ( f ) 368.1, 2 ( g ) 38.89, 2; ( h ) 108.3, 2); e two-way ANOVA with Bonferroni post hoc test ( F , df = 7.12, 2)

    Journal: BMC Medicine

    Article Title: Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

    doi: 10.1186/s12916-019-1423-5

    Figure Lengend Snippet: 5A-SM mobilizes cholesterol in vivo and ameliorates disease phenotypes . a Serum cholesterol from 7-week-old Npc1 I1061T homozygous mice pre- and 2 h post-treatment with 100 mg/kg 5A-SM, i.p. b Pre- (dashed line) and 2 h post-treatment (solid line) serum was fractionated by HPLC, and cholesterol was quantified by cholesterol oxidase assay. VLDL, LDL, and HDL fractions are indicated by arrows. c , d Seven-week-old wild-type (WT) and Npc1 I1061T homozygous (NPC) mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM. 48 h later, c liver HMGCS transcript levels and d total serum bilirubin were analyzed. e WT and Npc1 I1061T mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM three times per week from 7 to 11 weeks of age. The change in weight of each mouse from week 7 ( t = 0) to week 11 ( t = 4) was quantified. f Seven-week-old WT and NPC mice were injected with Veh or 100 mg/kg 5A-SM three times per week for 2 weeks. At 9 weeks of age livers were stained for macrophages using F4/80 (green) and DNA Hoechst (blue). Macrophage area is quantified at right. Scale bar = 50 μm. Violin plot shows median (dashed line), 25% and 75% (dotted lines), and probability density (thickness). g Brain slices from 8-week-old Npc1 I1061T mice were incubated with vehicle (Veh) or 5 mg/ml 5A-SM for 4 days, and filipin levels in Purkinje neuron soma were quantified (see also Additional file 1 : Figure S4b). h Six- to 7-week-old WT and Npc1 I1061T mice received intraventricular injections with vehicle (Veh) or 5A-SM-DiD. N: WT = 4, NPC Veh = 5, NPC 5A-SM = 4 mice. One-week later, cholesterol levels in Purkinje neuron soma (green) were analyzed by filipin (blue) staining. Dashed lines indicate Purkinje neuron soma (also see Additional file 1 : Figure S6a). Scale bar = 50 μm. Data quantified at right. Data are mean ± s.e.m. from a , b , c three; d genotype and treatment: number of mice, WT + Veh = 5, WT + 5A-SM = 3, NPC + Veh = 4, NPC + 5A-SM = 7; e genotype and treatment: number of mice at 9 weeks and 11 weeks, WT + Veh: 13 and 8, WT + 5A-SM: 9 and 8, NPC + Veh: 6, NPC + 5A-SM: 12 and 10 mice; f genotype and treatment: number of mice, cells, WT + Veh: 4, 301, NPC + Veh: 4, 514, NPC + 5A-SM: 3, 373 ( g ) WT = 93, NPC Veh = 143, NPC + 5A-SM = 116 cells. * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001. a , c Student’s t test ( t = ( a ) 6.375, ( c ) 5.23); d , f , g , h one-way ANOVA with Tukey post hoc test ( F , df = ( d ) 13.28, 3; ( f ) 368.1, 2 ( g ) 38.89, 2; ( h ) 108.3, 2); e two-way ANOVA with Bonferroni post hoc test ( F , df = 7.12, 2)

    Article Snippet: Filipin staining After treatment, cell membranes were labeled with wheat germ agglutinin® (Thermo Fisher).

    Techniques: In Vivo, Mouse Assay, High Performance Liquid Chromatography, Injection, Staining, Incubation

    sHDLs require ABCA1 to remove accumulated cholesterol from Niemann–Pick C fibroblasts. a – f Primary fibroblasts homozygous for NPC1 I1061T were treated with various sHDL formulations. a , b Accumulation of unesterified cholesterol was visualized by filipin staining ( a ) following 48-h treatment with increasing doses (representative images of 0.75 mg/ml) of vehicle (Veh), 5A peptide, 5A-POPC, 5A-SM, and 5A-DMPC (quantified below) or ( b ) with 0.75 mg/ml sHDL at various time points. c Effects of 48-h treatment with sHDL (0.75 mg/ml), 5A peptide, or vehicle (Veh) on total cellular cholesterol were measured using the Amplex Red assay. d The ratio of 5A or 22A peptide to sphingomyelin (SM) was altered during synthesis and the effect of peptide: SM ratio on cholesterol removal was determined by filipin staining (48-h treatment). e Cells were treated for two consecutive days with the following siRNAs: non-targeting (NT), ABCA1, or SR-B1, and concurrently treated with vehicle (Veh) or 5A-SM. Cholesterol storage was determined by filipin staining. f Cells were treated with cyclodextrin (Cyclo), 5A-SM, or 5A-SM preloaded with increasing amount of cholesterol content (5–20% total lipid weight), or human HDL (HuHDL). Cholesterol storage was assessed by filipin staining 48 h after treatment. Data are mean ± s.e.m. from ( a , b , e ) three, ( c ) five, ( d ) 5–8, or ( f ) 4–6 independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001 by a , b two-way ANOVA with Bonferroni post hoc test ( F , df = ( a ) 33.53, df = 4; ( b ) 32.88, 4), c – f one-way ANOVA with Tukey post hoc test ( F , df = ( c ) 13.98, 4; ( d ) 6.96, 8; ( e ) 22.5, 6; ( f ) 6.94, 5). a Dash lines indicate plasma membrane, scale bar = 20 μm

    Journal: BMC Medicine

    Article Title: Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

    doi: 10.1186/s12916-019-1423-5

    Figure Lengend Snippet: sHDLs require ABCA1 to remove accumulated cholesterol from Niemann–Pick C fibroblasts. a – f Primary fibroblasts homozygous for NPC1 I1061T were treated with various sHDL formulations. a , b Accumulation of unesterified cholesterol was visualized by filipin staining ( a ) following 48-h treatment with increasing doses (representative images of 0.75 mg/ml) of vehicle (Veh), 5A peptide, 5A-POPC, 5A-SM, and 5A-DMPC (quantified below) or ( b ) with 0.75 mg/ml sHDL at various time points. c Effects of 48-h treatment with sHDL (0.75 mg/ml), 5A peptide, or vehicle (Veh) on total cellular cholesterol were measured using the Amplex Red assay. d The ratio of 5A or 22A peptide to sphingomyelin (SM) was altered during synthesis and the effect of peptide: SM ratio on cholesterol removal was determined by filipin staining (48-h treatment). e Cells were treated for two consecutive days with the following siRNAs: non-targeting (NT), ABCA1, or SR-B1, and concurrently treated with vehicle (Veh) or 5A-SM. Cholesterol storage was determined by filipin staining. f Cells were treated with cyclodextrin (Cyclo), 5A-SM, or 5A-SM preloaded with increasing amount of cholesterol content (5–20% total lipid weight), or human HDL (HuHDL). Cholesterol storage was assessed by filipin staining 48 h after treatment. Data are mean ± s.e.m. from ( a , b , e ) three, ( c ) five, ( d ) 5–8, or ( f ) 4–6 independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001 by a , b two-way ANOVA with Bonferroni post hoc test ( F , df = ( a ) 33.53, df = 4; ( b ) 32.88, 4), c – f one-way ANOVA with Tukey post hoc test ( F , df = ( c ) 13.98, 4; ( d ) 6.96, 8; ( e ) 22.5, 6; ( f ) 6.94, 5). a Dash lines indicate plasma membrane, scale bar = 20 μm

    Article Snippet: Filipin staining After treatment, cell membranes were labeled with wheat germ agglutinin® (Thermo Fisher).

    Techniques: Staining, Amplex Red Assay

    5A-SM is endocytosed and increases cholesterol efflux. a – d NPC1 I1061T fibroblasts were treated with the indicated sHDL for a – c 2 or d 24 h. a Cells were pre-treated with dynasore (80 μM), amiloride (1 mM), or vehicle (Veh) for 30 min and then incubated with fresh media containing 5A-SM-DiD plus dynasore, amiloride, or vehicle for 2 h. Plasma membranes are outlined with dashed lines. 5A-SM-DiD (red) intensity is quantified at the right. b Cells were treated with sHDL composed of 5A-Alexa647 (green) and DiA (red) incorporated into the SM fraction. Following 2-h incubation, cells were labeled with NucStain (blue) and imaged by confocal microscopy. Pearson co-localization coefficient = 0.75 ± 0.01. c Cells were incubated with 5A-SM-DiD (red) for 1, 1.5, and 2 h, fixed, stained for LAMP1 (green) and filipin (blue), and imaged by confocal microscopy. Representative images from 2 h post-treatment. Pearson co-localization coefficient quantified below. d Cells were pre-treated for 24 h with acetylated LDL containing [3H] cholesteryl linoleate to specifically deliver cargo to the lysosomal compartment. Following 24-h equilibration, cells were treated for 24 h with 0.75 mg/ml 5A peptide or 5A-SM. Radioactivity in media and cell fractions was determined by liquid scintillation counting, and values were normalized to vehicle treated group. Data are mean ± s.e.m. from three independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, **** p ≤ .0001 by a one-way ANOVA with Tukey post hoc test relative to Veh or 5A ( F = 10.74, df = 2); c two-way ANOVA with Bonferroni post hoc test ( F , df = 23.63, 2). d Student’s t test t = 13.09, df = 4. Scale bar = a 12 μm, b 20 μm, c 10 μm

    Journal: BMC Medicine

    Article Title: Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

    doi: 10.1186/s12916-019-1423-5

    Figure Lengend Snippet: 5A-SM is endocytosed and increases cholesterol efflux. a – d NPC1 I1061T fibroblasts were treated with the indicated sHDL for a – c 2 or d 24 h. a Cells were pre-treated with dynasore (80 μM), amiloride (1 mM), or vehicle (Veh) for 30 min and then incubated with fresh media containing 5A-SM-DiD plus dynasore, amiloride, or vehicle for 2 h. Plasma membranes are outlined with dashed lines. 5A-SM-DiD (red) intensity is quantified at the right. b Cells were treated with sHDL composed of 5A-Alexa647 (green) and DiA (red) incorporated into the SM fraction. Following 2-h incubation, cells were labeled with NucStain (blue) and imaged by confocal microscopy. Pearson co-localization coefficient = 0.75 ± 0.01. c Cells were incubated with 5A-SM-DiD (red) for 1, 1.5, and 2 h, fixed, stained for LAMP1 (green) and filipin (blue), and imaged by confocal microscopy. Representative images from 2 h post-treatment. Pearson co-localization coefficient quantified below. d Cells were pre-treated for 24 h with acetylated LDL containing [3H] cholesteryl linoleate to specifically deliver cargo to the lysosomal compartment. Following 24-h equilibration, cells were treated for 24 h with 0.75 mg/ml 5A peptide or 5A-SM. Radioactivity in media and cell fractions was determined by liquid scintillation counting, and values were normalized to vehicle treated group. Data are mean ± s.e.m. from three independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, **** p ≤ .0001 by a one-way ANOVA with Tukey post hoc test relative to Veh or 5A ( F = 10.74, df = 2); c two-way ANOVA with Bonferroni post hoc test ( F , df = 23.63, 2). d Student’s t test t = 13.09, df = 4. Scale bar = a 12 μm, b 20 μm, c 10 μm

    Article Snippet: Filipin staining After treatment, cell membranes were labeled with wheat germ agglutinin® (Thermo Fisher).

    Techniques: Incubation, Labeling, Confocal Microscopy, Staining, Radioactivity

    5A-SM mobilizes cholesterol in vivo and ameliorates disease phenotypes . a Serum cholesterol from 7-week-old Npc1 I1061T homozygous mice pre- and 2 h post-treatment with 100 mg/kg 5A-SM, i.p. b Pre- (dashed line) and 2 h post-treatment (solid line) serum was fractionated by HPLC, and cholesterol was quantified by cholesterol oxidase assay. VLDL, LDL, and HDL fractions are indicated by arrows. c , d Seven-week-old wild-type (WT) and Npc1 I1061T homozygous (NPC) mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM. 48 h later, c liver HMGCS transcript levels and d total serum bilirubin were analyzed. e WT and Npc1 I1061T mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM three times per week from 7 to 11 weeks of age. The change in weight of each mouse from week 7 ( t = 0) to week 11 ( t = 4) was quantified. f Seven-week-old WT and NPC mice were injected with Veh or 100 mg/kg 5A-SM three times per week for 2 weeks. At 9 weeks of age livers were stained for macrophages using F4/80 (green) and DNA Hoechst (blue). Macrophage area is quantified at right. Scale bar = 50 μm. Violin plot shows median (dashed line), 25% and 75% (dotted lines), and probability density (thickness). g Brain slices from 8-week-old Npc1 I1061T mice were incubated with vehicle (Veh) or 5 mg/ml 5A-SM for 4 days, and filipin levels in Purkinje neuron soma were quantified (see also Additional file 1 : Figure S4b). h Six- to 7-week-old WT and Npc1 I1061T mice received intraventricular injections with vehicle (Veh) or 5A-SM-DiD. N: WT = 4, NPC Veh = 5, NPC 5A-SM = 4 mice. One-week later, cholesterol levels in Purkinje neuron soma (green) were analyzed by filipin (blue) staining. Dashed lines indicate Purkinje neuron soma (also see Additional file 1 : Figure S6a). Scale bar = 50 μm. Data quantified at right. Data are mean ± s.e.m. from a , b , c three; d genotype and treatment: number of mice, WT + Veh = 5, WT + 5A-SM = 3, NPC + Veh = 4, NPC + 5A-SM = 7; e genotype and treatment: number of mice at 9 weeks and 11 weeks, WT + Veh: 13 and 8, WT + 5A-SM: 9 and 8, NPC + Veh: 6, NPC + 5A-SM: 12 and 10 mice; f genotype and treatment: number of mice, cells, WT + Veh: 4, 301, NPC + Veh: 4, 514, NPC + 5A-SM: 3, 373 ( g ) WT = 93, NPC Veh = 143, NPC + 5A-SM = 116 cells. * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001. a , c Student’s t test ( t = ( a ) 6.375, ( c ) 5.23); d , f , g , h one-way ANOVA with Tukey post hoc test ( F , df = ( d ) 13.28, 3; ( f ) 368.1, 2 ( g ) 38.89, 2; ( h ) 108.3, 2); e two-way ANOVA with Bonferroni post hoc test ( F , df = 7.12, 2)

    Journal: BMC Medicine

    Article Title: Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

    doi: 10.1186/s12916-019-1423-5

    Figure Lengend Snippet: 5A-SM mobilizes cholesterol in vivo and ameliorates disease phenotypes . a Serum cholesterol from 7-week-old Npc1 I1061T homozygous mice pre- and 2 h post-treatment with 100 mg/kg 5A-SM, i.p. b Pre- (dashed line) and 2 h post-treatment (solid line) serum was fractionated by HPLC, and cholesterol was quantified by cholesterol oxidase assay. VLDL, LDL, and HDL fractions are indicated by arrows. c , d Seven-week-old wild-type (WT) and Npc1 I1061T homozygous (NPC) mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM. 48 h later, c liver HMGCS transcript levels and d total serum bilirubin were analyzed. e WT and Npc1 I1061T mice were injected i.p. with vehicle (Veh) or 100 mg/kg 5A-SM three times per week from 7 to 11 weeks of age. The change in weight of each mouse from week 7 ( t = 0) to week 11 ( t = 4) was quantified. f Seven-week-old WT and NPC mice were injected with Veh or 100 mg/kg 5A-SM three times per week for 2 weeks. At 9 weeks of age livers were stained for macrophages using F4/80 (green) and DNA Hoechst (blue). Macrophage area is quantified at right. Scale bar = 50 μm. Violin plot shows median (dashed line), 25% and 75% (dotted lines), and probability density (thickness). g Brain slices from 8-week-old Npc1 I1061T mice were incubated with vehicle (Veh) or 5 mg/ml 5A-SM for 4 days, and filipin levels in Purkinje neuron soma were quantified (see also Additional file 1 : Figure S4b). h Six- to 7-week-old WT and Npc1 I1061T mice received intraventricular injections with vehicle (Veh) or 5A-SM-DiD. N: WT = 4, NPC Veh = 5, NPC 5A-SM = 4 mice. One-week later, cholesterol levels in Purkinje neuron soma (green) were analyzed by filipin (blue) staining. Dashed lines indicate Purkinje neuron soma (also see Additional file 1 : Figure S6a). Scale bar = 50 μm. Data quantified at right. Data are mean ± s.e.m. from a , b , c three; d genotype and treatment: number of mice, WT + Veh = 5, WT + 5A-SM = 3, NPC + Veh = 4, NPC + 5A-SM = 7; e genotype and treatment: number of mice at 9 weeks and 11 weeks, WT + Veh: 13 and 8, WT + 5A-SM: 9 and 8, NPC + Veh: 6, NPC + 5A-SM: 12 and 10 mice; f genotype and treatment: number of mice, cells, WT + Veh: 4, 301, NPC + Veh: 4, 514, NPC + 5A-SM: 3, 373 ( g ) WT = 93, NPC Veh = 143, NPC + 5A-SM = 116 cells. * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001. a , c Student’s t test ( t = ( a ) 6.375, ( c ) 5.23); d , f , g , h one-way ANOVA with Tukey post hoc test ( F , df = ( d ) 13.28, 3; ( f ) 368.1, 2 ( g ) 38.89, 2; ( h ) 108.3, 2); e two-way ANOVA with Bonferroni post hoc test ( F , df = 7.12, 2)

    Article Snippet: The following day, slices were washed 3× in PBS and labeled with Alexa conjugated secondary (1:500) for 1 h. After 3 washes in PBS, slices were stained with filipin labeling solution for 2 h, washed 3× with PBS, and mounted in ProLong Gold (ThermoFisher) and imaged by confocal microscopy.

    Techniques: In Vivo, Mouse Assay, High Performance Liquid Chromatography, Injection, Staining, Incubation

    sHDLs require ABCA1 to remove accumulated cholesterol from Niemann–Pick C fibroblasts. a – f Primary fibroblasts homozygous for NPC1 I1061T were treated with various sHDL formulations. a , b Accumulation of unesterified cholesterol was visualized by filipin staining ( a ) following 48-h treatment with increasing doses (representative images of 0.75 mg/ml) of vehicle (Veh), 5A peptide, 5A-POPC, 5A-SM, and 5A-DMPC (quantified below) or ( b ) with 0.75 mg/ml sHDL at various time points. c Effects of 48-h treatment with sHDL (0.75 mg/ml), 5A peptide, or vehicle (Veh) on total cellular cholesterol were measured using the Amplex Red assay. d The ratio of 5A or 22A peptide to sphingomyelin (SM) was altered during synthesis and the effect of peptide: SM ratio on cholesterol removal was determined by filipin staining (48-h treatment). e Cells were treated for two consecutive days with the following siRNAs: non-targeting (NT), ABCA1, or SR-B1, and concurrently treated with vehicle (Veh) or 5A-SM. Cholesterol storage was determined by filipin staining. f Cells were treated with cyclodextrin (Cyclo), 5A-SM, or 5A-SM preloaded with increasing amount of cholesterol content (5–20% total lipid weight), or human HDL (HuHDL). Cholesterol storage was assessed by filipin staining 48 h after treatment. Data are mean ± s.e.m. from ( a , b , e ) three, ( c ) five, ( d ) 5–8, or ( f ) 4–6 independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001 by a , b two-way ANOVA with Bonferroni post hoc test ( F , df = ( a ) 33.53, df = 4; ( b ) 32.88, 4), c – f one-way ANOVA with Tukey post hoc test ( F , df = ( c ) 13.98, 4; ( d ) 6.96, 8; ( e ) 22.5, 6; ( f ) 6.94, 5). a Dash lines indicate plasma membrane, scale bar = 20 μm

    Journal: BMC Medicine

    Article Title: Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

    doi: 10.1186/s12916-019-1423-5

    Figure Lengend Snippet: sHDLs require ABCA1 to remove accumulated cholesterol from Niemann–Pick C fibroblasts. a – f Primary fibroblasts homozygous for NPC1 I1061T were treated with various sHDL formulations. a , b Accumulation of unesterified cholesterol was visualized by filipin staining ( a ) following 48-h treatment with increasing doses (representative images of 0.75 mg/ml) of vehicle (Veh), 5A peptide, 5A-POPC, 5A-SM, and 5A-DMPC (quantified below) or ( b ) with 0.75 mg/ml sHDL at various time points. c Effects of 48-h treatment with sHDL (0.75 mg/ml), 5A peptide, or vehicle (Veh) on total cellular cholesterol were measured using the Amplex Red assay. d The ratio of 5A or 22A peptide to sphingomyelin (SM) was altered during synthesis and the effect of peptide: SM ratio on cholesterol removal was determined by filipin staining (48-h treatment). e Cells were treated for two consecutive days with the following siRNAs: non-targeting (NT), ABCA1, or SR-B1, and concurrently treated with vehicle (Veh) or 5A-SM. Cholesterol storage was determined by filipin staining. f Cells were treated with cyclodextrin (Cyclo), 5A-SM, or 5A-SM preloaded with increasing amount of cholesterol content (5–20% total lipid weight), or human HDL (HuHDL). Cholesterol storage was assessed by filipin staining 48 h after treatment. Data are mean ± s.e.m. from ( a , b , e ) three, ( c ) five, ( d ) 5–8, or ( f ) 4–6 independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, *** p ≤ .001, **** p ≤ .0001 by a , b two-way ANOVA with Bonferroni post hoc test ( F , df = ( a ) 33.53, df = 4; ( b ) 32.88, 4), c – f one-way ANOVA with Tukey post hoc test ( F , df = ( c ) 13.98, 4; ( d ) 6.96, 8; ( e ) 22.5, 6; ( f ) 6.94, 5). a Dash lines indicate plasma membrane, scale bar = 20 μm

    Article Snippet: The following day, slices were washed 3× in PBS and labeled with Alexa conjugated secondary (1:500) for 1 h. After 3 washes in PBS, slices were stained with filipin labeling solution for 2 h, washed 3× with PBS, and mounted in ProLong Gold (ThermoFisher) and imaged by confocal microscopy.

    Techniques: Staining, Amplex Red Assay

    5A-SM is endocytosed and increases cholesterol efflux. a – d NPC1 I1061T fibroblasts were treated with the indicated sHDL for a – c 2 or d 24 h. a Cells were pre-treated with dynasore (80 μM), amiloride (1 mM), or vehicle (Veh) for 30 min and then incubated with fresh media containing 5A-SM-DiD plus dynasore, amiloride, or vehicle for 2 h. Plasma membranes are outlined with dashed lines. 5A-SM-DiD (red) intensity is quantified at the right. b Cells were treated with sHDL composed of 5A-Alexa647 (green) and DiA (red) incorporated into the SM fraction. Following 2-h incubation, cells were labeled with NucStain (blue) and imaged by confocal microscopy. Pearson co-localization coefficient = 0.75 ± 0.01. c Cells were incubated with 5A-SM-DiD (red) for 1, 1.5, and 2 h, fixed, stained for LAMP1 (green) and filipin (blue), and imaged by confocal microscopy. Representative images from 2 h post-treatment. Pearson co-localization coefficient quantified below. d Cells were pre-treated for 24 h with acetylated LDL containing [3H] cholesteryl linoleate to specifically deliver cargo to the lysosomal compartment. Following 24-h equilibration, cells were treated for 24 h with 0.75 mg/ml 5A peptide or 5A-SM. Radioactivity in media and cell fractions was determined by liquid scintillation counting, and values were normalized to vehicle treated group. Data are mean ± s.e.m. from three independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, **** p ≤ .0001 by a one-way ANOVA with Tukey post hoc test relative to Veh or 5A ( F = 10.74, df = 2); c two-way ANOVA with Bonferroni post hoc test ( F , df = 23.63, 2). d Student’s t test t = 13.09, df = 4. Scale bar = a 12 μm, b 20 μm, c 10 μm

    Journal: BMC Medicine

    Article Title: Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

    doi: 10.1186/s12916-019-1423-5

    Figure Lengend Snippet: 5A-SM is endocytosed and increases cholesterol efflux. a – d NPC1 I1061T fibroblasts were treated with the indicated sHDL for a – c 2 or d 24 h. a Cells were pre-treated with dynasore (80 μM), amiloride (1 mM), or vehicle (Veh) for 30 min and then incubated with fresh media containing 5A-SM-DiD plus dynasore, amiloride, or vehicle for 2 h. Plasma membranes are outlined with dashed lines. 5A-SM-DiD (red) intensity is quantified at the right. b Cells were treated with sHDL composed of 5A-Alexa647 (green) and DiA (red) incorporated into the SM fraction. Following 2-h incubation, cells were labeled with NucStain (blue) and imaged by confocal microscopy. Pearson co-localization coefficient = 0.75 ± 0.01. c Cells were incubated with 5A-SM-DiD (red) for 1, 1.5, and 2 h, fixed, stained for LAMP1 (green) and filipin (blue), and imaged by confocal microscopy. Representative images from 2 h post-treatment. Pearson co-localization coefficient quantified below. d Cells were pre-treated for 24 h with acetylated LDL containing [3H] cholesteryl linoleate to specifically deliver cargo to the lysosomal compartment. Following 24-h equilibration, cells were treated for 24 h with 0.75 mg/ml 5A peptide or 5A-SM. Radioactivity in media and cell fractions was determined by liquid scintillation counting, and values were normalized to vehicle treated group. Data are mean ± s.e.m. from three independent experiments. n.s., not significant, * p ≤ .05, ** p ≤ .01, **** p ≤ .0001 by a one-way ANOVA with Tukey post hoc test relative to Veh or 5A ( F = 10.74, df = 2); c two-way ANOVA with Bonferroni post hoc test ( F , df = 23.63, 2). d Student’s t test t = 13.09, df = 4. Scale bar = a 12 μm, b 20 μm, c 10 μm

    Article Snippet: The following day, slices were washed 3× in PBS and labeled with Alexa conjugated secondary (1:500) for 1 h. After 3 washes in PBS, slices were stained with filipin labeling solution for 2 h, washed 3× with PBS, and mounted in ProLong Gold (ThermoFisher) and imaged by confocal microscopy.

    Techniques: Incubation, Labeling, Confocal Microscopy, Staining, Radioactivity