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
Filipin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/filipin/product/Thermo Fisher
Average 93 stars, based on 12 article reviews
Price from $9.99 to $1999.99
filipin - by Bioz Stars, 2020-09
93/100 stars

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 "BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts"

Article Title: BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200404106

Attenuation of BDNF enhancement of synaptic exocytosis by lipid raft disruption. Recycling synaptic vesicles in cultured hippocampal or cortical neurons were labeled by FM1-43, and exocytosis was induced by a perfusion of 50 mM KCl containing KRH buffer (high K + solution). (A) Pseudo-colored images showing high K + solution-dependent reduction in FM1-43 intensity. FM 1-43 images were captured at the indicated times (s) and baseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min before stimulation. Arrowheads indicate representative spots with a gradual reduction of FM dye labeling after depolarization. Bar, 10 μm. (B) Representative recordings of FM 1-43 destaining. Neurons were incubated with or without BDNF for 30 min, followed by a 1-min exposure to high K + solution to load FM dye. After three washes for 5 min, FM dye was destained with high K + solution (black arrow). FM 1-43 intensities at 4 s before (gray arrow) and at 15 s after (white arrow) depolarization are defined as F b and F a , respectively. ΔF (%) = (F b −F a )/F b × 100. (C–E) Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells were pretreated with MCD, MCD–cholesterol complex (MCD-Chol), or filipin for 10 min, followed by BDNF incubation for 30 min and stimulation with high K + solution. Data were collected at 4 s before and at 15 s after stimulation with high K + solution. The number associated with each column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in the intensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.
Figure Legend Snippet: Attenuation of BDNF enhancement of synaptic exocytosis by lipid raft disruption. Recycling synaptic vesicles in cultured hippocampal or cortical neurons were labeled by FM1-43, and exocytosis was induced by a perfusion of 50 mM KCl containing KRH buffer (high K + solution). (A) Pseudo-colored images showing high K + solution-dependent reduction in FM1-43 intensity. FM 1-43 images were captured at the indicated times (s) and baseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min before stimulation. Arrowheads indicate representative spots with a gradual reduction of FM dye labeling after depolarization. Bar, 10 μm. (B) Representative recordings of FM 1-43 destaining. Neurons were incubated with or without BDNF for 30 min, followed by a 1-min exposure to high K + solution to load FM dye. After three washes for 5 min, FM dye was destained with high K + solution (black arrow). FM 1-43 intensities at 4 s before (gray arrow) and at 15 s after (white arrow) depolarization are defined as F b and F a , respectively. ΔF (%) = (F b −F a )/F b × 100. (C–E) Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells were pretreated with MCD, MCD–cholesterol complex (MCD-Chol), or filipin for 10 min, followed by BDNF incubation for 30 min and stimulation with high K + solution. Data were collected at 4 s before and at 15 s after stimulation with high K + solution. The number associated with each column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in the intensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.

Techniques Used: Cell Culture, Labeling, Incubation

4) Product Images from "Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth"

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

Journal: Cancer letters

doi: 10.1016/j.canlet.2017.09.009

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Microscopy, Staining, AST Assay

5) 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

6) 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

7) Product Images from "Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells"

Article Title: Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells

Journal: bioRxiv

doi: 10.1101/076695

Quantification of effects of Vorinostat on cholesterol accumulation in NPC1 mutants. The effect of 10 μM Vorinostat was tested on 81 different NPC1 mutations from five segments of the NPC1 protein as illustrated in Figure 8 . DMSO was used as a solvent control. Filipin fluorescence images of the transfected cells were analyzed to obtain an LSO value as explained in methods. Data represent averages ± SEM from 15-25 images. Each image includes about 1-5 transfected cells. Dark blue (DMSO treated) and light blue bars (Vorinostat treated) for each group represent mutants that showed reduction in LSO values with p
Figure Legend Snippet: Quantification of effects of Vorinostat on cholesterol accumulation in NPC1 mutants. The effect of 10 μM Vorinostat was tested on 81 different NPC1 mutations from five segments of the NPC1 protein as illustrated in Figure 8 . DMSO was used as a solvent control. Filipin fluorescence images of the transfected cells were analyzed to obtain an LSO value as explained in methods. Data represent averages ± SEM from 15-25 images. Each image includes about 1-5 transfected cells. Dark blue (DMSO treated) and light blue bars (Vorinostat treated) for each group represent mutants that showed reduction in LSO values with p

Techniques Used: Fluorescence, Transfection

Dose dependent effect of Vorinostat and Panobinostat on multiple patient derived NPC1 mutant cell lines. NPC1 mutant human fibroblasts were treated with Vorinostat (A) or Panobinostat (B) for 48 hours followed by fixation, staining with filipin and imaging using the ImageXpress Micro automatic fluorescence microscope. DMSO was used as a solvent control. Images were analyzed to obtain the LSO value as a measure of cholesterol accumulation. Data were normalized to the corresponding DMSO treated cells. Data for each cell line are averages of three independent experiments totaling 60 images (5 wells x 4 sites x 3 experiments). Error bars: SEM.
Figure Legend Snippet: Dose dependent effect of Vorinostat and Panobinostat on multiple patient derived NPC1 mutant cell lines. NPC1 mutant human fibroblasts were treated with Vorinostat (A) or Panobinostat (B) for 48 hours followed by fixation, staining with filipin and imaging using the ImageXpress Micro automatic fluorescence microscope. DMSO was used as a solvent control. Images were analyzed to obtain the LSO value as a measure of cholesterol accumulation. Data were normalized to the corresponding DMSO treated cells. Data for each cell line are averages of three independent experiments totaling 60 images (5 wells x 4 sites x 3 experiments). Error bars: SEM.

Techniques Used: Derivative Assay, Mutagenesis, Staining, Imaging, Fluorescence, Microscopy

Illustration of the screening system. A. Representative images of transfected cells. 24 hours after transfection with a bicistronic vector containing eGFP plus wild type NPC1 or NPC1 I1061T , U2OS-SRA-shNPC1 cells were treated with Vorinostat (10 µM), Panobinostat (50 nM), or DMSO solvent control for 48 hours. Cells were treated with 50 µg/ml AcLDL for the final 2 hours, fixed with PFA, and stained with filipin. Images were acquired on a Leica wide-field microscope using standard GFP and A4 filters. Transfection with wild type NPC1 reduces the cholesterol accumulation, but transfection with NPC1 I1061T does not unless they are treated with an HDACi. B. Quantification of filipin in transfected cells. Cholesterol accumulation in LSO of GFP positive cells was measured based on filipin intensity, and the LSO values are shown. Error bars: SEM.
Figure Legend Snippet: Illustration of the screening system. A. Representative images of transfected cells. 24 hours after transfection with a bicistronic vector containing eGFP plus wild type NPC1 or NPC1 I1061T , U2OS-SRA-shNPC1 cells were treated with Vorinostat (10 µM), Panobinostat (50 nM), or DMSO solvent control for 48 hours. Cells were treated with 50 µg/ml AcLDL for the final 2 hours, fixed with PFA, and stained with filipin. Images were acquired on a Leica wide-field microscope using standard GFP and A4 filters. Transfection with wild type NPC1 reduces the cholesterol accumulation, but transfection with NPC1 I1061T does not unless they are treated with an HDACi. B. Quantification of filipin in transfected cells. Cholesterol accumulation in LSO of GFP positive cells was measured based on filipin intensity, and the LSO values are shown. Error bars: SEM.

Techniques Used: Transfection, Plasmid Preparation, Staining, Microscopy

Persistent effect of HDACi treatment in NPC1 mutant human fibroblasts. GM03123 ( A ) or GM18453 ( B ) cells were treated with Vorinostat at varying concentrations for 72 hours. The cells were then incubated for an additional 0, 1, 2 or 3 days without Vorinostat in normal growth medium. At the end of each time point cells were stained with filipin and the LSO value was measured. Data for each cell line are from two independent experiments, and each data point is obtained using 48 images. Each data point is normalized to its corresponding DMSO treated condition, so the value of one represents no effect. Error bars: SEM.
Figure Legend Snippet: Persistent effect of HDACi treatment in NPC1 mutant human fibroblasts. GM03123 ( A ) or GM18453 ( B ) cells were treated with Vorinostat at varying concentrations for 72 hours. The cells were then incubated for an additional 0, 1, 2 or 3 days without Vorinostat in normal growth medium. At the end of each time point cells were stained with filipin and the LSO value was measured. Data for each cell line are from two independent experiments, and each data point is obtained using 48 images. Each data point is normalized to its corresponding DMSO treated condition, so the value of one represents no effect. Error bars: SEM.

Techniques Used: Mutagenesis, Incubation, Staining

8) 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

9) Product Images from "Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment"

Article Title: Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.00580

Effect of filipin on IL-6 and IL-8 secretion by A549 cells during interaction with H. capsulatum . A549 cells were incubated in the absence or presence of 1 μg/ml filipin (FIL, a cholesterol-binding compound that disrupts membrane rafts). After 2 h, H. capsulatum yeasts (Hc) were added to these cultures. After 16 h, culture supernatants were collected, and IL-6 (A) and IL-8 (B) levels were determined by ELISA. To analyze basal cytokine levels, A549 cells were incubated in the absence of filipin and H. capsulatum (FIL -/Hc -).Values represent the mean of triplicate experiments ± the standard deviation. ∗ p
Figure Legend Snippet: Effect of filipin on IL-6 and IL-8 secretion by A549 cells during interaction with H. capsulatum . A549 cells were incubated in the absence or presence of 1 μg/ml filipin (FIL, a cholesterol-binding compound that disrupts membrane rafts). After 2 h, H. capsulatum yeasts (Hc) were added to these cultures. After 16 h, culture supernatants were collected, and IL-6 (A) and IL-8 (B) levels were determined by ELISA. To analyze basal cytokine levels, A549 cells were incubated in the absence of filipin and H. capsulatum (FIL -/Hc -).Values represent the mean of triplicate experiments ± the standard deviation. ∗ p

Techniques Used: Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

10) 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

11) 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

12) 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

13) 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

14) Product Images from "Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles"

Article Title: Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200409068

Enzymatic modification of regulatory lipids inhibits vertex assembly. Docking reactions (30 min, 27°C), using vacuoles containing GFP-Ypt7p (A) or vacuoles labeled with Cy3-FYVE to mark PI(3)P (B) or filipin to mark ergosterol (C), bore 1 μM MTM-1, 2 μM SigD, 1 U/ml PI-PLC, or 2.7 μM Plc1p. After incubation, reactions were placed on ice, counterstained with FM4-64 (A and C) or MDY-64 (B), and prepared for microscopic analysis. Geometric means ± 95% confidence intervals of the relative vertex enrichment are shown.
Figure Legend Snippet: Enzymatic modification of regulatory lipids inhibits vertex assembly. Docking reactions (30 min, 27°C), using vacuoles containing GFP-Ypt7p (A) or vacuoles labeled with Cy3-FYVE to mark PI(3)P (B) or filipin to mark ergosterol (C), bore 1 μM MTM-1, 2 μM SigD, 1 U/ml PI-PLC, or 2.7 μM Plc1p. After incubation, reactions were placed on ice, counterstained with FM4-64 (A and C) or MDY-64 (B), and prepared for microscopic analysis. Geometric means ± 95% confidence intervals of the relative vertex enrichment are shown.

Techniques Used: Modification, Labeling, Planar Chromatography, Incubation

Regulatory lipids are required for vacuolar association of Vam7p. Vam7p association with vacuolar membranes was assayed by immunoblot in fusion reactions without inhibitor or with 2 μM GST-FYVE, 10 μM C1b, 19 μM filipin, 38 μM GST-ENTH, 2 μM SigD, or 1 μM MTM-1. Reactions were also performed in the presence of anti-Sec18p F ab to inhibit priming. Fusion reactions were incubated for 90 min at 27°C, then fractionated into membrane pellets and supernatants by centrifugation (13,000 g , 15 min, 4°C). Membranes were resuspended in 30 μl PS buffer with protease inhibitors (1 μM leupeptin, 5 μM pepstatin and 0.1 μM pefabloc-SC). (A) Equal proportions of the pellet and supernatant fractions were mixed with SDS-loading buffer, resolved by SDS-PAGE, then transferred to nitrocellulose. (B and C) Quantitative analysis of membrane-bound Vam7p by Western blots. Data represent mean values ± SEM. Relative band intensities were measured using NIH Image 1.62 ( n = 4).
Figure Legend Snippet: Regulatory lipids are required for vacuolar association of Vam7p. Vam7p association with vacuolar membranes was assayed by immunoblot in fusion reactions without inhibitor or with 2 μM GST-FYVE, 10 μM C1b, 19 μM filipin, 38 μM GST-ENTH, 2 μM SigD, or 1 μM MTM-1. Reactions were also performed in the presence of anti-Sec18p F ab to inhibit priming. Fusion reactions were incubated for 90 min at 27°C, then fractionated into membrane pellets and supernatants by centrifugation (13,000 g , 15 min, 4°C). Membranes were resuspended in 30 μl PS buffer with protease inhibitors (1 μM leupeptin, 5 μM pepstatin and 0.1 μM pefabloc-SC). (A) Equal proportions of the pellet and supernatant fractions were mixed with SDS-loading buffer, resolved by SDS-PAGE, then transferred to nitrocellulose. (B and C) Quantitative analysis of membrane-bound Vam7p by Western blots. Data represent mean values ± SEM. Relative band intensities were measured using NIH Image 1.62 ( n = 4).

Techniques Used: Incubation, Centrifugation, SDS Page, Western Blot

Regulatory lipids are not required for Ypt7p-dependent tethering. For quantitative microscopic assay of docking ( Mayer and Wickner, 1997 ), vacuoles were incubated with PS buffer or treated with 2.8 μM Gdi1p and 11.4 μM Gyp1-46p for 10 min at 27°C in the absence of ATP. Aliquots were then added to chilled tubes containing docking buffer, 2 μM GST-FYVE, 10 μM MED, 30 μM ENTH, 10 μM C1b or 19 μM filipin. Reactions were supplemented with the docking reaction ATP-regenerating system and returned to 27°C for 20 min. After incubation, docking reactions were placed on ice, incubated for 2 min with FM4-64 and mounted on slides for analysis. For each condition, 10 random fields were scored for cluster size. Vacuole clusters (A, black bars) include some vacuoles of enlarged diameter, reflecting fusion (not depicted).
Figure Legend Snippet: Regulatory lipids are not required for Ypt7p-dependent tethering. For quantitative microscopic assay of docking ( Mayer and Wickner, 1997 ), vacuoles were incubated with PS buffer or treated with 2.8 μM Gdi1p and 11.4 μM Gyp1-46p for 10 min at 27°C in the absence of ATP. Aliquots were then added to chilled tubes containing docking buffer, 2 μM GST-FYVE, 10 μM MED, 30 μM ENTH, 10 μM C1b or 19 μM filipin. Reactions were supplemented with the docking reaction ATP-regenerating system and returned to 27°C for 20 min. After incubation, docking reactions were placed on ice, incubated for 2 min with FM4-64 and mounted on slides for analysis. For each condition, 10 random fields were scored for cluster size. Vacuole clusters (A, black bars) include some vacuoles of enlarged diameter, reflecting fusion (not depicted).

Techniques Used: Incubation

Regulatory lipids control the vertex enrichment of Ypt7p, SNAREs, and HOPS. Docking reactions using vacuoles from strains expressing GFP fusions to Ypt7p (A), Vam7p (B), Vam3p (C), Vti1p (D), Vps33p (E), or Pho8p (F) were treated with 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or 2 μM GST-FYVE and assayed for vertex enrichment. Reactions were incubated for 30 min at 27°C, placed on ice and labeled with FM4-64. Geometric mean values ± 95% confidence intervals of relative vertex enrichment are shown. (G–R) Fluorescent images of docked vacuoles containing GFP-Vti1p (G–L), or GFP-Ypt7p (M–R). Docking reactions bore no inhibitor (G–I and M–O) or 30 μM ENTH (J–L and P–R). G, J, M, and P show membrane staining with FM4-64. H, K, N, and Q show the distribution of GFP-Vti1p (H and K) or GFP-Ypt7p (N and Q). Merged images illustrate the enrichment of GFP-Vti1p (I) and Ypt7p (O) at vertices relative to outer membrane. ENTH treatment abolished the vertex enrichment of these proteins (L and R). Arrows are examples of vertices enriched in GFP-Vti1 (H and I) and GFP-Ypt7 (N and O) relative to outer membrane. Bars, 5 μm.
Figure Legend Snippet: Regulatory lipids control the vertex enrichment of Ypt7p, SNAREs, and HOPS. Docking reactions using vacuoles from strains expressing GFP fusions to Ypt7p (A), Vam7p (B), Vam3p (C), Vti1p (D), Vps33p (E), or Pho8p (F) were treated with 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or 2 μM GST-FYVE and assayed for vertex enrichment. Reactions were incubated for 30 min at 27°C, placed on ice and labeled with FM4-64. Geometric mean values ± 95% confidence intervals of relative vertex enrichment are shown. (G–R) Fluorescent images of docked vacuoles containing GFP-Vti1p (G–L), or GFP-Ypt7p (M–R). Docking reactions bore no inhibitor (G–I and M–O) or 30 μM ENTH (J–L and P–R). G, J, M, and P show membrane staining with FM4-64. H, K, N, and Q show the distribution of GFP-Vti1p (H and K) or GFP-Ypt7p (N and Q). Merged images illustrate the enrichment of GFP-Vti1p (I) and Ypt7p (O) at vertices relative to outer membrane. ENTH treatment abolished the vertex enrichment of these proteins (L and R). Arrows are examples of vertices enriched in GFP-Vti1 (H and I) and GFP-Ypt7 (N and O) relative to outer membrane. Bars, 5 μm.

Techniques Used: Expressing, Incubation, Labeling, Staining

Interdependence of the vertex enrichment of regulatory lipids. Docking reactions were labeled with (A) 0.2 μM Cy3-FYVE or (B) 5 μM filipin and incubated with either 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or buffer alone. After 30 min at 27°C, reactions were placed on ice, labeled with MDY-64 (A) or FM4-64 (B), and prepared for fluorescence microscopy. Relative enrichments of specific probes were determined as in Fig. 3 . Data are presented as geometric mean values ± 95% confidence intervals of the relative enrichment at vertices.
Figure Legend Snippet: Interdependence of the vertex enrichment of regulatory lipids. Docking reactions were labeled with (A) 0.2 μM Cy3-FYVE or (B) 5 μM filipin and incubated with either 30 μM ENTH, 10 μM MED, 10 μM C1b, 19 μM filipin, 25 μM PX, or buffer alone. After 30 min at 27°C, reactions were placed on ice, labeled with MDY-64 (A) or FM4-64 (B), and prepared for fluorescence microscopy. Relative enrichments of specific probes were determined as in Fig. 3 . Data are presented as geometric mean values ± 95% confidence intervals of the relative enrichment at vertices.

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy

Lipids are enriched at the vertices of docked vacuoles. Vacuoles from DKY6281 yeast were incubated under docking conditions for 30 min at 27°C with fluorescent lipid ligands. After incubation, docking reactions were placed on ice and labeled with either FM4-64 or MDY-64. (A) Outer membrane, boundary membrane and vertex ring subdomains of docked vacuoles. (B and E) MDY-64 or (H, K, and N) FM4-64 label the entire membrane. Specific lipids were labeled with (C) 0.2 μM Cy3-FYVE to label PI(3)P, (F) 0.6 μM Cy3-ENTH to label PI(4,5)P 2 , (I) 5 μM filipin to label ergosterol, (L) 1 μM Alexa488-C1b to label DAG, or (O) 2.5 μM PSS-380 to label PS. For Cy3-ENTH labeling, vacuoles were reisolated (5,220 g , 4°C, 5 min) at the end of the 30-min incubation to eliminate background fluorescence from unbound probe and resuspended in the original volume of PS buffer before labeling with MDY-64 and analysis. D, G, J, M, and P show merged images of nonspecific and specific probes. Closed arrows are examples of vertex sites enriched in the specified regulatory lipid. Open arrows are outer membrane microdomains. Bars, 2 μm. As shown below ( Fig. 9 ), ENTH displaces Vam7p from membranes after 90 min under fusion conditions which are different from the docking conditions and time (30 min) used here.
Figure Legend Snippet: Lipids are enriched at the vertices of docked vacuoles. Vacuoles from DKY6281 yeast were incubated under docking conditions for 30 min at 27°C with fluorescent lipid ligands. After incubation, docking reactions were placed on ice and labeled with either FM4-64 or MDY-64. (A) Outer membrane, boundary membrane and vertex ring subdomains of docked vacuoles. (B and E) MDY-64 or (H, K, and N) FM4-64 label the entire membrane. Specific lipids were labeled with (C) 0.2 μM Cy3-FYVE to label PI(3)P, (F) 0.6 μM Cy3-ENTH to label PI(4,5)P 2 , (I) 5 μM filipin to label ergosterol, (L) 1 μM Alexa488-C1b to label DAG, or (O) 2.5 μM PSS-380 to label PS. For Cy3-ENTH labeling, vacuoles were reisolated (5,220 g , 4°C, 5 min) at the end of the 30-min incubation to eliminate background fluorescence from unbound probe and resuspended in the original volume of PS buffer before labeling with MDY-64 and analysis. D, G, J, M, and P show merged images of nonspecific and specific probes. Closed arrows are examples of vertex sites enriched in the specified regulatory lipid. Open arrows are outer membrane microdomains. Bars, 2 μm. As shown below ( Fig. 9 ), ENTH displaces Vam7p from membranes after 90 min under fusion conditions which are different from the docking conditions and time (30 min) used here.

Techniques Used: Incubation, Labeling, Fluorescence

Ligands and enzymes which target phosphoinositides, DAG and ergosterol inhibit vacuole fusion. Fusion reactions with vacuoles from BJ3505 and DKY6281 were performed in the absence or presence of the indicated concentrations of GST-FYVE (A), MED (B), ENTH (C), C1b (D), Filipin (E), PSS-380 (F), MTM-1 (G), or SigD (H), added from the start of the reaction. Fusion was assayed by phosphatase activity and expressed in U. Closed arrows indicate inhibitory concentrations used in this study. Open arrows indicate sub-inhibitory concentrations used for determining lipid localization.
Figure Legend Snippet: Ligands and enzymes which target phosphoinositides, DAG and ergosterol inhibit vacuole fusion. Fusion reactions with vacuoles from BJ3505 and DKY6281 were performed in the absence or presence of the indicated concentrations of GST-FYVE (A), MED (B), ENTH (C), C1b (D), Filipin (E), PSS-380 (F), MTM-1 (G), or SigD (H), added from the start of the reaction. Fusion was assayed by phosphatase activity and expressed in U. Closed arrows indicate inhibitory concentrations used in this study. Open arrows indicate sub-inhibitory concentrations used for determining lipid localization.

Techniques Used: Activity Assay

15) Product Images from "Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment"

Article Title: Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment

Journal: Cellular Microbiology

doi: 10.1111/cmi.12279

Intracellular cholesterol sources colocalize with ML in foamy macrophages of LL patients.A. Sections of skin biopsies of LL patients ( n = 4) were analysed showing colocalization between LD organelles (green) and ML (red).B. High magnification of the boxed areas visualized in (A) showing bacteria completely enveloped by LDs (yellow arrow).C–H. Virchow's cells isolated from LL biopsies were stained with different fluorophores and anti-ADRP to visualize Cho sources within these ML-infected foamy macrophages. (C) Oil red O reveals the foamy phenotype of the isolated Virchow's cell. (D) Fluorescence and (E) Differential interference contrast (DIC) of a Virchow's cell showing ML residing in Cho-rich regions (yellow arrow). (F) LD–ML association within macrophages isolated from LL biopsies was visualized using BODIPY label. (G) Immunofluorescence confocal images for ADRP (green) and anti-LAM (red) show the LD–ML association (yellow arrow) (H) LDL-[BODIPY-Cho] internalization in Virchow's cells and intimate association with ML (yellow arrow). Nuclei were stained with TO-PRO-3. No fluorescence was observed with the isotype control IgG (Supplementary Fig. S2 ). Bars: white = 10 mm; yellow = 5 mm. Filipin was used as a Cho label (blue). BODIPY and ADRP were used as LD marker (green); LDL-Cho: LDL fluorescent as LDL-[BODIPY-Cho] (green), LAM (lipoarabinomannan) was used as an ML marker (red).
Figure Legend Snippet: Intracellular cholesterol sources colocalize with ML in foamy macrophages of LL patients.A. Sections of skin biopsies of LL patients ( n = 4) were analysed showing colocalization between LD organelles (green) and ML (red).B. High magnification of the boxed areas visualized in (A) showing bacteria completely enveloped by LDs (yellow arrow).C–H. Virchow's cells isolated from LL biopsies were stained with different fluorophores and anti-ADRP to visualize Cho sources within these ML-infected foamy macrophages. (C) Oil red O reveals the foamy phenotype of the isolated Virchow's cell. (D) Fluorescence and (E) Differential interference contrast (DIC) of a Virchow's cell showing ML residing in Cho-rich regions (yellow arrow). (F) LD–ML association within macrophages isolated from LL biopsies was visualized using BODIPY label. (G) Immunofluorescence confocal images for ADRP (green) and anti-LAM (red) show the LD–ML association (yellow arrow) (H) LDL-[BODIPY-Cho] internalization in Virchow's cells and intimate association with ML (yellow arrow). Nuclei were stained with TO-PRO-3. No fluorescence was observed with the isotype control IgG (Supplementary Fig. S2 ). Bars: white = 10 mm; yellow = 5 mm. Filipin was used as a Cho label (blue). BODIPY and ADRP were used as LD marker (green); LDL-Cho: LDL fluorescent as LDL-[BODIPY-Cho] (green), LAM (lipoarabinomannan) was used as an ML marker (red).

Techniques Used: Isolation, Staining, Infection, Fluorescence, Immunofluorescence, Laser Capture Microdissection, Marker

Cellular incorporation of LDL-cholesterol in ML-infected macrophages. Forty-eight hours after infection in medium containing 2% FCS, infected macrophages were pulse-labelled at 33°C with LDL-Cho for 2 h in medium serum free.A. The effect of native LDL-Cho and ML infection on intracellular Cho levels measured by filipin staining and flow cytometry.B. In parallel, the quantitative analysis of total Cho was determined by Amplex Red Cholesterol kit under the same conditions.C. Cells were treated with dead or live bacteria in the same condition described above and the LDL-[BODIPY-Cho] incorporation was determined by flow cytometry.D. To validate the fluorescence assay the LDL-[ 3 H]-Cho incorporation was assayed under same condition of fluorescence assay.C–E. The internalization of LDL-[BODIPY-Cho] as a process dependent on bacterial viability (C and D) and multiplicity of infection (moi) (E) as determined by flow cytometry.F. Bacterial association and LDL-[BODIPY-Cho] were measured simultaneously by flow cytometry. A separate analysis was performed in cells with no bacteria (cells without internalized ML) and cells bearing bacteria (cells with internalized ML). MFI values of LDL-[BODIPY-Cho] are expressed in bar graphs.Results from five representative experiments are shown. *Statistically significant differences ( P ≤ 0.05) when comparing ML-infected cells with control cells. +Statistically significant differences ( P ≤ 0.05) when comparing different ML-treated cell groups; n.s: non-significant.G and H. Fluorescence images of macrophage cultures showing LDL-[BODIPY-Cho] in cells associated or not with ML. (G) Macrophages isolated from PBMC and (H) Virchow cells isolated from LL skin biopsies. Original bar, 10 μm.
Figure Legend Snippet: Cellular incorporation of LDL-cholesterol in ML-infected macrophages. Forty-eight hours after infection in medium containing 2% FCS, infected macrophages were pulse-labelled at 33°C with LDL-Cho for 2 h in medium serum free.A. The effect of native LDL-Cho and ML infection on intracellular Cho levels measured by filipin staining and flow cytometry.B. In parallel, the quantitative analysis of total Cho was determined by Amplex Red Cholesterol kit under the same conditions.C. Cells were treated with dead or live bacteria in the same condition described above and the LDL-[BODIPY-Cho] incorporation was determined by flow cytometry.D. To validate the fluorescence assay the LDL-[ 3 H]-Cho incorporation was assayed under same condition of fluorescence assay.C–E. The internalization of LDL-[BODIPY-Cho] as a process dependent on bacterial viability (C and D) and multiplicity of infection (moi) (E) as determined by flow cytometry.F. Bacterial association and LDL-[BODIPY-Cho] were measured simultaneously by flow cytometry. A separate analysis was performed in cells with no bacteria (cells without internalized ML) and cells bearing bacteria (cells with internalized ML). MFI values of LDL-[BODIPY-Cho] are expressed in bar graphs.Results from five representative experiments are shown. *Statistically significant differences ( P ≤ 0.05) when comparing ML-infected cells with control cells. +Statistically significant differences ( P ≤ 0.05) when comparing different ML-treated cell groups; n.s: non-significant.G and H. Fluorescence images of macrophage cultures showing LDL-[BODIPY-Cho] in cells associated or not with ML. (G) Macrophages isolated from PBMC and (H) Virchow cells isolated from LL skin biopsies. Original bar, 10 μm.

Techniques Used: Infection, Staining, Flow Cytometry, Cytometry, Fluorescence, Isolation

Lipid accumulation in vivo and in vitro in ML-infected tissues and cells.A. Sections of skin biopsies from BT and LL patients ( n = 4) were immunostained for ADRP and observed by immunofluorescence confocal microscopy. Bar: 20 μm (yellow).B. Total RNA was extracted from BT and LL biopsies, and expression of ADRP was normalized to GADPH and quantified by qRT-PCR. Values are the average of four independent experiments.C. Representative HPTLC of neutral lipids extracted from skin biopsies of BT and LL patients detected by charring.D. The content of each class of lipids was estimated by densitometry and plotted as a percentage.E. Lipid accumulation in uninfected and ML-infected macrophages was observed by immunostaining for ADRP. Bar: 10 μm (white).F. LD formation was measured by Nile Red staining.G and H. (G) Representative HPTLC of neutral lipids extracted from macrophages treated or not with ML and (H) the percentage of each class of lipids estimated by densitometry.I. Quantitative Cho levels estimated by filipin staining and flow cytometry.In (A) and (E) Nuclei (blue) were labelled with DAPI. No fluorescence was observed for the Alexa546-labelled mouse isotype control IgG. In HPTLC, the position of pure standard lipids is indicated on the margin of the panel. *Statistically significant differences ( P ≤ 0.05) comparing LL groups, BT groups, and ML-infected cells versus uninfected cells.
Figure Legend Snippet: Lipid accumulation in vivo and in vitro in ML-infected tissues and cells.A. Sections of skin biopsies from BT and LL patients ( n = 4) were immunostained for ADRP and observed by immunofluorescence confocal microscopy. Bar: 20 μm (yellow).B. Total RNA was extracted from BT and LL biopsies, and expression of ADRP was normalized to GADPH and quantified by qRT-PCR. Values are the average of four independent experiments.C. Representative HPTLC of neutral lipids extracted from skin biopsies of BT and LL patients detected by charring.D. The content of each class of lipids was estimated by densitometry and plotted as a percentage.E. Lipid accumulation in uninfected and ML-infected macrophages was observed by immunostaining for ADRP. Bar: 10 μm (white).F. LD formation was measured by Nile Red staining.G and H. (G) Representative HPTLC of neutral lipids extracted from macrophages treated or not with ML and (H) the percentage of each class of lipids estimated by densitometry.I. Quantitative Cho levels estimated by filipin staining and flow cytometry.In (A) and (E) Nuclei (blue) were labelled with DAPI. No fluorescence was observed for the Alexa546-labelled mouse isotype control IgG. In HPTLC, the position of pure standard lipids is indicated on the margin of the panel. *Statistically significant differences ( P ≤ 0.05) comparing LL groups, BT groups, and ML-infected cells versus uninfected cells.

Techniques Used: In Vivo, In Vitro, Infection, Immunofluorescence, Confocal Microscopy, Expressing, Quantitative RT-PCR, High Performance Thin Layer Chromatography, Immunostaining, Staining, Flow Cytometry, Cytometry, Fluorescence

16) 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

17) 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

18) 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

Microscopy:

Article Title: Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth
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). .. HUVEC were seeded in a Nunc Lab-Tek II 8-Chamber Slide and treated with compounds for indicated time points.

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. ..

Staining:

Article Title: Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth
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). .. HUVEC were seeded in a Nunc Lab-Tek II 8-Chamber Slide and treated with compounds for indicated time points.

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: 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: BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts
Article Snippet: .. After washing three times with KRH buffer, neurons were pretreated with or without MCD, MCD–cholesterol complex or filipin for 10 min followed by BDNF incubation for 30 min. FM dye (2 mM; Molecular Probes) was loaded by incubating the cells with the high K+ solution (KRH buffer containing 56 mM KCl and 79 mM NaCl) for 1 min at 37°C. .. After washing the cells with KRH buffer, fluorescence images of FM-labeled spots were taken every second using fluorescence microscope (model IX70; Olympus) equipped with a Cool SNAP HQ CCD camera (Roper Scientific) and a 20 × 0.8 NA objective (Olympus).

Labeling:

Article Title: Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells
Article Snippet: .. All reagent additions, buffer changes and labeling with filipin were carried out robotically using a Thermo Multi-Drop liquid dispenser and a Bio-Tek Elx405 plate washer (Bio-Tek Instruments, Inc., Winooski, VT). .. Fluorescence Microscopy An automated ImageXpressMicro imaging system from Molecular Devices equipped with a 300W Xenon-arc lamp from Perkin-Elmer, a Nikon 10X Plan Fluor 0.3 numerical aperture (NA) objective, and a Photometrics CoolSnapHQ camera (1,392 x 1,040 pixels) from Roper Scientific was used to acquire images.

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. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    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: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin/product/Thermo Fisher
    Average 93 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    filipin - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    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: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin staining/product/Thermo Fisher
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    filipin staining - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    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

    Quantification of effects of Vorinostat on cholesterol accumulation in NPC1 mutants. The effect of 10 μM Vorinostat was tested on 81 different NPC1 mutations from five segments of the NPC1 protein as illustrated in Figure 8 . DMSO was used as a solvent control. Filipin fluorescence images of the transfected cells were analyzed to obtain an LSO value as explained in methods. Data represent averages ± SEM from 15-25 images. Each image includes about 1-5 transfected cells. Dark blue (DMSO treated) and light blue bars (Vorinostat treated) for each group represent mutants that showed reduction in LSO values with p

    Journal: bioRxiv

    Article Title: Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells

    doi: 10.1101/076695

    Figure Lengend Snippet: Quantification of effects of Vorinostat on cholesterol accumulation in NPC1 mutants. The effect of 10 μM Vorinostat was tested on 81 different NPC1 mutations from five segments of the NPC1 protein as illustrated in Figure 8 . DMSO was used as a solvent control. Filipin fluorescence images of the transfected cells were analyzed to obtain an LSO value as explained in methods. Data represent averages ± SEM from 15-25 images. Each image includes about 1-5 transfected cells. Dark blue (DMSO treated) and light blue bars (Vorinostat treated) for each group represent mutants that showed reduction in LSO values with p

    Article Snippet: All reagent additions, buffer changes and labeling with filipin were carried out robotically using a Thermo Multi-Drop liquid dispenser and a Bio-Tek Elx405 plate washer (Bio-Tek Instruments, Inc., Winooski, VT).

    Techniques: Fluorescence, Transfection

    Dose dependent effect of Vorinostat and Panobinostat on multiple patient derived NPC1 mutant cell lines. NPC1 mutant human fibroblasts were treated with Vorinostat (A) or Panobinostat (B) for 48 hours followed by fixation, staining with filipin and imaging using the ImageXpress Micro automatic fluorescence microscope. DMSO was used as a solvent control. Images were analyzed to obtain the LSO value as a measure of cholesterol accumulation. Data were normalized to the corresponding DMSO treated cells. Data for each cell line are averages of three independent experiments totaling 60 images (5 wells x 4 sites x 3 experiments). Error bars: SEM.

    Journal: bioRxiv

    Article Title: Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells

    doi: 10.1101/076695

    Figure Lengend Snippet: Dose dependent effect of Vorinostat and Panobinostat on multiple patient derived NPC1 mutant cell lines. NPC1 mutant human fibroblasts were treated with Vorinostat (A) or Panobinostat (B) for 48 hours followed by fixation, staining with filipin and imaging using the ImageXpress Micro automatic fluorescence microscope. DMSO was used as a solvent control. Images were analyzed to obtain the LSO value as a measure of cholesterol accumulation. Data were normalized to the corresponding DMSO treated cells. Data for each cell line are averages of three independent experiments totaling 60 images (5 wells x 4 sites x 3 experiments). Error bars: SEM.

    Article Snippet: All reagent additions, buffer changes and labeling with filipin were carried out robotically using a Thermo Multi-Drop liquid dispenser and a Bio-Tek Elx405 plate washer (Bio-Tek Instruments, Inc., Winooski, VT).

    Techniques: Derivative Assay, Mutagenesis, Staining, Imaging, Fluorescence, Microscopy

    Illustration of the screening system. A. Representative images of transfected cells. 24 hours after transfection with a bicistronic vector containing eGFP plus wild type NPC1 or NPC1 I1061T , U2OS-SRA-shNPC1 cells were treated with Vorinostat (10 µM), Panobinostat (50 nM), or DMSO solvent control for 48 hours. Cells were treated with 50 µg/ml AcLDL for the final 2 hours, fixed with PFA, and stained with filipin. Images were acquired on a Leica wide-field microscope using standard GFP and A4 filters. Transfection with wild type NPC1 reduces the cholesterol accumulation, but transfection with NPC1 I1061T does not unless they are treated with an HDACi. B. Quantification of filipin in transfected cells. Cholesterol accumulation in LSO of GFP positive cells was measured based on filipin intensity, and the LSO values are shown. Error bars: SEM.

    Journal: bioRxiv

    Article Title: Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells

    doi: 10.1101/076695

    Figure Lengend Snippet: Illustration of the screening system. A. Representative images of transfected cells. 24 hours after transfection with a bicistronic vector containing eGFP plus wild type NPC1 or NPC1 I1061T , U2OS-SRA-shNPC1 cells were treated with Vorinostat (10 µM), Panobinostat (50 nM), or DMSO solvent control for 48 hours. Cells were treated with 50 µg/ml AcLDL for the final 2 hours, fixed with PFA, and stained with filipin. Images were acquired on a Leica wide-field microscope using standard GFP and A4 filters. Transfection with wild type NPC1 reduces the cholesterol accumulation, but transfection with NPC1 I1061T does not unless they are treated with an HDACi. B. Quantification of filipin in transfected cells. Cholesterol accumulation in LSO of GFP positive cells was measured based on filipin intensity, and the LSO values are shown. Error bars: SEM.

    Article Snippet: All reagent additions, buffer changes and labeling with filipin were carried out robotically using a Thermo Multi-Drop liquid dispenser and a Bio-Tek Elx405 plate washer (Bio-Tek Instruments, Inc., Winooski, VT).

    Techniques: Transfection, Plasmid Preparation, Staining, Microscopy

    Persistent effect of HDACi treatment in NPC1 mutant human fibroblasts. GM03123 ( A ) or GM18453 ( B ) cells were treated with Vorinostat at varying concentrations for 72 hours. The cells were then incubated for an additional 0, 1, 2 or 3 days without Vorinostat in normal growth medium. At the end of each time point cells were stained with filipin and the LSO value was measured. Data for each cell line are from two independent experiments, and each data point is obtained using 48 images. Each data point is normalized to its corresponding DMSO treated condition, so the value of one represents no effect. Error bars: SEM.

    Journal: bioRxiv

    Article Title: Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells

    doi: 10.1101/076695

    Figure Lengend Snippet: Persistent effect of HDACi treatment in NPC1 mutant human fibroblasts. GM03123 ( A ) or GM18453 ( B ) cells were treated with Vorinostat at varying concentrations for 72 hours. The cells were then incubated for an additional 0, 1, 2 or 3 days without Vorinostat in normal growth medium. At the end of each time point cells were stained with filipin and the LSO value was measured. Data for each cell line are from two independent experiments, and each data point is obtained using 48 images. Each data point is normalized to its corresponding DMSO treated condition, so the value of one represents no effect. Error bars: SEM.

    Article Snippet: All reagent additions, buffer changes and labeling with filipin were carried out robotically using a Thermo Multi-Drop liquid dispenser and a Bio-Tek Elx405 plate washer (Bio-Tek Instruments, Inc., Winooski, VT).

    Techniques: Mutagenesis, Incubation, Staining

    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