Structured Review

Polysciences inc filipin
Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by <t>filipin</t> fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P
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1) Product Images from "PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport"

Article Title: PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074169

Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by filipin fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P
Figure Legend Snippet: Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by filipin fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P

Techniques Used: Activation Assay, Fluorescence

Effects of fatty acids on vimentin solubilization and the NPC1 phenotype. (A) Human NPC1 3123 cells were treated with 50µg/ml linoleic or oleic acid for 24 hrs, after which the levels of soluble vimentin were analyzed by Western blotting. Cholesterol storage in NPC1 CHO (B) or human 3123 (C) cells was analyzed by filipin staining. Fluorescence intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. * and *** denote statistically significant differences between treated and untreated cells with P
Figure Legend Snippet: Effects of fatty acids on vimentin solubilization and the NPC1 phenotype. (A) Human NPC1 3123 cells were treated with 50µg/ml linoleic or oleic acid for 24 hrs, after which the levels of soluble vimentin were analyzed by Western blotting. Cholesterol storage in NPC1 CHO (B) or human 3123 (C) cells was analyzed by filipin staining. Fluorescence intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. * and *** denote statistically significant differences between treated and untreated cells with P

Techniques Used: Western Blot, Staining, Fluorescence

Effects of transient PKC expression on the NPC1 phenotype. M12 cells were transfected with PKC isoforms or Rab9 for 48 hrs and then analyzed by filipin staining for cholesterol storage. Cells positive for transfection stain positive for GFP (left panel) and show decreased filipin staining (outlined cells, right panel) compared to surrounding untransfected cells, confirming the role of PKCs in mobilizing stored cholesterol from the NPC1 endosomes. Bar, 20μ.
Figure Legend Snippet: Effects of transient PKC expression on the NPC1 phenotype. M12 cells were transfected with PKC isoforms or Rab9 for 48 hrs and then analyzed by filipin staining for cholesterol storage. Cells positive for transfection stain positive for GFP (left panel) and show decreased filipin staining (outlined cells, right panel) compared to surrounding untransfected cells, confirming the role of PKCs in mobilizing stored cholesterol from the NPC1 endosomes. Bar, 20μ.

Techniques Used: Expressing, Transfection, Staining

2) Product Images from "An "Exacerbate-reverse" Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease *"

Article Title: An "Exacerbate-reverse" Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.227645

Global histone deacetylase inhibition corrects cholesterol homeostasis in NP-C fibroblasts. Mutant fibroblasts (NPC-26 and NPC-J4) were incubated for 18 h in the presence of 5 μ m SAHA, stained with filipin, and assessed for esterification of LDL-cholesterol.
Figure Legend Snippet: Global histone deacetylase inhibition corrects cholesterol homeostasis in NP-C fibroblasts. Mutant fibroblasts (NPC-26 and NPC-J4) were incubated for 18 h in the presence of 5 μ m SAHA, stained with filipin, and assessed for esterification of LDL-cholesterol.

Techniques Used: Histone Deacetylase Assay, Inhibition, Mutagenesis, Incubation, Staining

3) Product Images from "Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds"

Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.034165

Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.
Figure Legend Snippet: Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

Techniques Used: Mutagenesis, Staining, Injection

2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P
Figure Legend Snippet: 2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

Techniques Used: Staining, Mutagenesis

Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.
Figure Legend Snippet: Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

Techniques Used: Expressing, Injection, Staining, Mutagenesis

4) Product Images from "Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease"

Article Title: Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease

Journal: Scientific Reports

doi: 10.1038/srep04356

Lysosomal cholesterol reduction ability of biocleavable HE-SS-PRX against NPC1 cells. (A) Filipin staining for unesterified cholesterol and LysoTracker staining for endosome/lysosome in normal and NPC1 cells (bar: 20 μm). The NPC1 cells were incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at the β-CD concentration of 0.1 mM for 24 h. (B) The amount of intracellular total cholesterol in NPC1 cells incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at various β-CD concentrations for 24 h. The dashed lines represent the amount of intracellular total cholesterol in non-treated normal and NPC1 cells. (C) Time-course of the amount of intracellular total cholesterol in NPC1 cells after incubation with HP-β-CD (β-CD: 10 mM), DM-β-CD (β-CD: 1 mM), non-cleavable HE-PRX (β-CD: 10 mM), and cleavable HE-SS-PRX (β-CD: 0.2 mM) for 24 h, followed by incubation without treatment for the indicated time periods. The closed circles represent the non-treated NPC1 cells. Data are expressed as the mean ± S.D. (n = 3) (* p
Figure Legend Snippet: Lysosomal cholesterol reduction ability of biocleavable HE-SS-PRX against NPC1 cells. (A) Filipin staining for unesterified cholesterol and LysoTracker staining for endosome/lysosome in normal and NPC1 cells (bar: 20 μm). The NPC1 cells were incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at the β-CD concentration of 0.1 mM for 24 h. (B) The amount of intracellular total cholesterol in NPC1 cells incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at various β-CD concentrations for 24 h. The dashed lines represent the amount of intracellular total cholesterol in non-treated normal and NPC1 cells. (C) Time-course of the amount of intracellular total cholesterol in NPC1 cells after incubation with HP-β-CD (β-CD: 10 mM), DM-β-CD (β-CD: 1 mM), non-cleavable HE-PRX (β-CD: 10 mM), and cleavable HE-SS-PRX (β-CD: 0.2 mM) for 24 h, followed by incubation without treatment for the indicated time periods. The closed circles represent the non-treated NPC1 cells. Data are expressed as the mean ± S.D. (n = 3) (* p

Techniques Used: Staining, Incubation, Concentration Assay

5) Product Images from "Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells"

Article Title: Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI15420

Overexpression of WT Rab7 or Rab9 (but not Rab11) in NP-C cells reduced the accumulation of intracellular cholesterol. ( a ) Cells were transfected with plasmids expressing EGFP fusion proteins of WT Rab7, Rab9, or Rab11 and cultured in EMEM plus 10% FBS. Forty-eight hours later, the distribution of free cholesterol was examined by filipin staining (blue fluorescence). Transfected cells identified by green fluorescence are outlined in the filipin images. Bar, 25 μm. ( b ) Quantitation of filipin fluorescence in NP-C cells overexpressing WT Rab7, Rab9, or Rab11 proteins. Total filipin fluorescence of individual transfected or adjacent untransfected cells was calculated by image analysis. Values shown are mean ± SD from a typical experiment ( n = 50). Similar results were obtained in each of three independent experiments.
Figure Legend Snippet: Overexpression of WT Rab7 or Rab9 (but not Rab11) in NP-C cells reduced the accumulation of intracellular cholesterol. ( a ) Cells were transfected with plasmids expressing EGFP fusion proteins of WT Rab7, Rab9, or Rab11 and cultured in EMEM plus 10% FBS. Forty-eight hours later, the distribution of free cholesterol was examined by filipin staining (blue fluorescence). Transfected cells identified by green fluorescence are outlined in the filipin images. Bar, 25 μm. ( b ) Quantitation of filipin fluorescence in NP-C cells overexpressing WT Rab7, Rab9, or Rab11 proteins. Total filipin fluorescence of individual transfected or adjacent untransfected cells was calculated by image analysis. Values shown are mean ± SD from a typical experiment ( n = 50). Similar results were obtained in each of three independent experiments.

Techniques Used: Over Expression, Transfection, Expressing, Cell Culture, Staining, Fluorescence, Quantitation Assay

6) Product Images from "Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole"

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole

Journal: eLife

doi: 10.7554/eLife.25960

Analysis of MVB and vps4Δ . ( A ) Freeze-fracture EM of MVB in ypt7Δ . ILVs in the lumen are observed as rugged structures (arrows). Bar: 0.2 μm. ( B ) Filipin staining in vps4Δ after nitrogen starvation. Vph1p–GFP (green) was observed in the vacuolar membrane (arrowheads) and in the class E compartment (arrows). Filipin staining was intense in the class E compartment, but it was negligible in the vacuolar membrane area. Bar: 5 μm. ( C ) Distribution of NPC proteins in vps4Δ after nitrogen starvation. Ncr1p–GFP and Npc2p–GFP were observed in the vacuolar membrane and in the vacuolar lumen, respectively. Bar: 5 μm. ( D ) The vacuolar content of atg7Δpep4Δprb1Δ and atg7Δ after nitrogen starvation. The vacuolar lumen of atg7Δpep4Δprb1Δ after nitrogen starvation contained a large number of ILVs, whereas that of atg7Δ was virtually vacant. Bar: 0.2 μm. ( E ) vps4Δ in stationary phase. ( a ) Domain formation was completely absent in vps4Δ . See Figure 3A for comparison. Mean ± SD of three independent experiments ( > 50 vacuoles were counted for each group in each experiment) are represented. ( b ) Lipophagy was also absent in vps4Δ . See Figure 3B for comparison. Mean ± SD of one representative experiment (n > 150 for each group) out of two repeats are shown. **p
Figure Legend Snippet: Analysis of MVB and vps4Δ . ( A ) Freeze-fracture EM of MVB in ypt7Δ . ILVs in the lumen are observed as rugged structures (arrows). Bar: 0.2 μm. ( B ) Filipin staining in vps4Δ after nitrogen starvation. Vph1p–GFP (green) was observed in the vacuolar membrane (arrowheads) and in the class E compartment (arrows). Filipin staining was intense in the class E compartment, but it was negligible in the vacuolar membrane area. Bar: 5 μm. ( C ) Distribution of NPC proteins in vps4Δ after nitrogen starvation. Ncr1p–GFP and Npc2p–GFP were observed in the vacuolar membrane and in the vacuolar lumen, respectively. Bar: 5 μm. ( D ) The vacuolar content of atg7Δpep4Δprb1Δ and atg7Δ after nitrogen starvation. The vacuolar lumen of atg7Δpep4Δprb1Δ after nitrogen starvation contained a large number of ILVs, whereas that of atg7Δ was virtually vacant. Bar: 0.2 μm. ( E ) vps4Δ in stationary phase. ( a ) Domain formation was completely absent in vps4Δ . See Figure 3A for comparison. Mean ± SD of three independent experiments ( > 50 vacuoles were counted for each group in each experiment) are represented. ( b ) Lipophagy was also absent in vps4Δ . See Figure 3B for comparison. Mean ± SD of one representative experiment (n > 150 for each group) out of two repeats are shown. **p

Techniques Used: Staining

Filipin staining. ( A ) Wild-type yeast in log phase was labeled only in the cell surface, but in stationary phase, it showed intense labeling in the vacuolar membrane as well as in the cell surface (arrows), giving rise to a double-ring staining pattern. Bar: 5 μm. ( B ) The vacuolar raft-like domain in the stationary phase WT yeast showed membrane deformation caused by filipin–sterol complexes (arrows). Bar: 0.2 μm. DOI: http://dx.doi.org/10.7554/eLife.25960.011
Figure Legend Snippet: Filipin staining. ( A ) Wild-type yeast in log phase was labeled only in the cell surface, but in stationary phase, it showed intense labeling in the vacuolar membrane as well as in the cell surface (arrows), giving rise to a double-ring staining pattern. Bar: 5 μm. ( B ) The vacuolar raft-like domain in the stationary phase WT yeast showed membrane deformation caused by filipin–sterol complexes (arrows). Bar: 0.2 μm. DOI: http://dx.doi.org/10.7554/eLife.25960.011

Techniques Used: Staining, Labeling

Filipin staining of atg7Δ in stationary phase. Filipin staining was performed by the method described for Figure 3E , and the proportion of cells showing the double-ring filipin-staining pattern was measured. The ratio was significantly lower in atg7Δ than in WT, indicating that the sterol concentration in the vacuolar membrane of atg7Δ was less than that of WT. Bar: 5 μm. **p
Figure Legend Snippet: Filipin staining of atg7Δ in stationary phase. Filipin staining was performed by the method described for Figure 3E , and the proportion of cells showing the double-ring filipin-staining pattern was measured. The ratio was significantly lower in atg7Δ than in WT, indicating that the sterol concentration in the vacuolar membrane of atg7Δ was less than that of WT. Bar: 5 μm. **p

Techniques Used: Staining, Concentration Assay

7) Product Images from "Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization"

Article Title: Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization

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

doi:

CT60 cell transfected with the C-4 NPC1 mutant lacking the dileucine motif. The mutant NPC1 protein ( A ) is present in a reticular pattern characteristic of the endoplasmic reticulum and in the nuclear envelope ( B ). The cell expressing mutant NPC1 has not cleared of cholesterol evidenced by its accumulation of filipin-positive lysosomes similar to the adjacent cell, not expressing mutant NPC1 protein. (Bar = 5 μm.)
Figure Legend Snippet: CT60 cell transfected with the C-4 NPC1 mutant lacking the dileucine motif. The mutant NPC1 protein ( A ) is present in a reticular pattern characteristic of the endoplasmic reticulum and in the nuclear envelope ( B ). The cell expressing mutant NPC1 has not cleared of cholesterol evidenced by its accumulation of filipin-positive lysosomes similar to the adjacent cell, not expressing mutant NPC1 protein. (Bar = 5 μm.)

Techniques Used: Transfection, Mutagenesis, Expressing

CT60 cell transfected with the C97S NPC1 mutant. ( A ) Filipin staining showed transfected cells did not clear of cholesterol and that the NPC1 protein ( B ) was present in rings at the surface of the cholesterol-laden cores of lysosomes that are lgp95 positive ( C ). The lgp95 immunostaining that marks protein in the lysosomal membrane colocalizes with the mutant NPC1 protein, indicating that the mutant NPC1 is at the surface of lysosomes. D is the merged image of A and B ; and E is the merged image of B and C . (Bar = 2.5 μm.)
Figure Legend Snippet: CT60 cell transfected with the C97S NPC1 mutant. ( A ) Filipin staining showed transfected cells did not clear of cholesterol and that the NPC1 protein ( B ) was present in rings at the surface of the cholesterol-laden cores of lysosomes that are lgp95 positive ( C ). The lgp95 immunostaining that marks protein in the lysosomal membrane colocalizes with the mutant NPC1 protein, indicating that the mutant NPC1 is at the surface of lysosomes. D is the merged image of A and B ; and E is the merged image of B and C . (Bar = 2.5 μm.)

Techniques Used: Transfection, Mutagenesis, Staining, Immunostaining

Photomicrographs of filipin-stained CT60 cells transfected with empty vector or wild-type NPC1 expression plasmid. ( A ) EGFP expression by CT60 cells transfected with pEGFP and empty vector. ( B ) Filipin staining. Arrows indicate EGFP-expressing cells. ( C ) EGFP expression by CT60 cells transfected with pEGFP and wild-type NPC1 expression plasmid. ( D ) Filipin staining. Arrows indicate EGFP-expressing cells. (Bar = 10 μm.)
Figure Legend Snippet: Photomicrographs of filipin-stained CT60 cells transfected with empty vector or wild-type NPC1 expression plasmid. ( A ) EGFP expression by CT60 cells transfected with pEGFP and empty vector. ( B ) Filipin staining. Arrows indicate EGFP-expressing cells. ( C ) EGFP expression by CT60 cells transfected with pEGFP and wild-type NPC1 expression plasmid. ( D ) Filipin staining. Arrows indicate EGFP-expressing cells. (Bar = 10 μm.)

Techniques Used: Staining, Transfection, Plasmid Preparation, Expressing

8) Product Images from "Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds"

Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.034165

Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.
Figure Legend Snippet: Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

Techniques Used: Mutagenesis, Staining, Injection

2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P
Figure Legend Snippet: 2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

Techniques Used: Staining, Mutagenesis

Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.
Figure Legend Snippet: Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

Techniques Used: Expressing, Injection, Staining, Mutagenesis

9) Product Images from "Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1"

Article Title: Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddw367

Effects of AAV9-EF1a-NPC1 vs AAV9-CamKII-NPC1 gene therapy on the liver of Npc1 -/- mice. Immunohistochemical imaging of cholesterol (filipin, A–D ) and Kupffer cells (CD68+, E–H ). Npc1 +/+ mice (A,E) exhibit no cholesterol accumulation or filipin staining (A) and Kupffer cells are minimal (E) whereas Npc1 -/- mice without treatment (B,F) have extensive cholesterol accumulation (B) and increased abundance of Kupffer cells (F). AAV9-EF1a-NPC1 treated Npc1 -/- mice (C,G) have hepatocytes free of cholesterol storage (C; e.g. white arrows) but demonstrate cholesterol storage in Kupffer cells (G), while AAV9-CamKII-NPC1 treated Npc1 -/- mice show no correction of either cell type (D,H). Scale bar = 50 μm. ( I ) Western blot of liver homogenate from control and treated mice showing NPC1 protein (∼180 kDa) in the untreated Npc1 +/+ and an AAV9-EF1a-NPC1 treated Npc1 -/- mouse with Tubulin (50 kDa) serving as loading control.
Figure Legend Snippet: Effects of AAV9-EF1a-NPC1 vs AAV9-CamKII-NPC1 gene therapy on the liver of Npc1 -/- mice. Immunohistochemical imaging of cholesterol (filipin, A–D ) and Kupffer cells (CD68+, E–H ). Npc1 +/+ mice (A,E) exhibit no cholesterol accumulation or filipin staining (A) and Kupffer cells are minimal (E) whereas Npc1 -/- mice without treatment (B,F) have extensive cholesterol accumulation (B) and increased abundance of Kupffer cells (F). AAV9-EF1a-NPC1 treated Npc1 -/- mice (C,G) have hepatocytes free of cholesterol storage (C; e.g. white arrows) but demonstrate cholesterol storage in Kupffer cells (G), while AAV9-CamKII-NPC1 treated Npc1 -/- mice show no correction of either cell type (D,H). Scale bar = 50 μm. ( I ) Western blot of liver homogenate from control and treated mice showing NPC1 protein (∼180 kDa) in the untreated Npc1 +/+ and an AAV9-EF1a-NPC1 treated Npc1 -/- mouse with Tubulin (50 kDa) serving as loading control.

Techniques Used: Mouse Assay, Immunohistochemistry, Imaging, Staining, Western Blot

Effect of AAV9-CamKII-NPC1 treatment in the CA3 hippocampus and layer V neocortex of Npc1 -/- mice. (A–R) Immunohistochemical imaging of mouse Npc1 or human NPC1 protein (red) in the hippocampus and Layer V neocortex, co-stained with NeuN (green) and filipin (blue). ( A–F ) Endogenous Npc1 expression in the Npc1 +/+ mouse, with NeuN stain removed in (B,D,F) to better show the neuronal Npc1 or NPC1 expression and magnified images of Layer V neocortex (C,D) and CA3 hippocampus (E,F). (G–L) Endogenous expression of Npc1 or NPC1 protein in the Npc1 -/- mouse, with NeuN stain removed in (H,J,L) to better show the lack of Npc1 or NPC1 and high level of intracellular filipin inclusions, with magnified images of Layer V neocortex (I,J) and CA3 hippocampus (K,L). (M-R) NPC1 expression in the Npc1 -/- mice injected with AAV9-CamKII - NPC1. NeuN stain removed in (N,P,R) to better show the presence of NPC1 in some neurons and the reduced level of intracellular filipin inclusions, with magnified images of Layer V neocortex (O,P), and CA3 hippocampus (Q,R). Quantification of filipin and Npc1 or NPC1 mean pixel intensity of the neuronal cell body in Layer V neocortical neurons (S, T) and CA3 hippocampal neurons (U, V), data expressed as mean ± S.E.M. A.U. = arbitrary units. * P
Figure Legend Snippet: Effect of AAV9-CamKII-NPC1 treatment in the CA3 hippocampus and layer V neocortex of Npc1 -/- mice. (A–R) Immunohistochemical imaging of mouse Npc1 or human NPC1 protein (red) in the hippocampus and Layer V neocortex, co-stained with NeuN (green) and filipin (blue). ( A–F ) Endogenous Npc1 expression in the Npc1 +/+ mouse, with NeuN stain removed in (B,D,F) to better show the neuronal Npc1 or NPC1 expression and magnified images of Layer V neocortex (C,D) and CA3 hippocampus (E,F). (G–L) Endogenous expression of Npc1 or NPC1 protein in the Npc1 -/- mouse, with NeuN stain removed in (H,J,L) to better show the lack of Npc1 or NPC1 and high level of intracellular filipin inclusions, with magnified images of Layer V neocortex (I,J) and CA3 hippocampus (K,L). (M-R) NPC1 expression in the Npc1 -/- mice injected with AAV9-CamKII - NPC1. NeuN stain removed in (N,P,R) to better show the presence of NPC1 in some neurons and the reduced level of intracellular filipin inclusions, with magnified images of Layer V neocortex (O,P), and CA3 hippocampus (Q,R). Quantification of filipin and Npc1 or NPC1 mean pixel intensity of the neuronal cell body in Layer V neocortical neurons (S, T) and CA3 hippocampal neurons (U, V), data expressed as mean ± S.E.M. A.U. = arbitrary units. * P

Techniques Used: Mouse Assay, Immunohistochemistry, Imaging, Staining, Expressing, Injection

Biochemical correction of the cholesterol storage phenotype in neurons transduced with AAV9-CamKII-NPC1 in the Npc1 -/- mouse. Immunohistochemical imaging of NPC1 protein levels (red) in Layer V neocortex (A,B) and CA3 of hippocampus ( E,F ), co-stained with filipin (blue) and NeuN (green, in A and E only). Arrows indicate neurons without appreciable NPC1 protein and high filipin staining. Arrowheads indicate neurons successfully transduced by the AAV9-CamKII-NPC1 with subsequent intense NPC1 staining and reduced filipin labelling. ( C,D ) NPC1 intensity of all Layer V neurons measured, plotted against filipin intensity. ( G,H ) NPC1 intensity of all CA3 hippocampal neurons measured, plotted against filipin intensity. Upper left quadrants in (D,H) indicate the percentage of Npc1 -/- neurons corrected to control levels with AAV9-CamKII-NPC1 treatment. Imaged density of AAV-CamKII-GFP ( I,J ) and AAV9-CamKII-NPC1 ( K,L ) incorporation in the Layer V cortex ( I,K ) and CA3 hippocampus ( J,L ), with quantification in ( M,N ). ns = non-significant.
Figure Legend Snippet: Biochemical correction of the cholesterol storage phenotype in neurons transduced with AAV9-CamKII-NPC1 in the Npc1 -/- mouse. Immunohistochemical imaging of NPC1 protein levels (red) in Layer V neocortex (A,B) and CA3 of hippocampus ( E,F ), co-stained with filipin (blue) and NeuN (green, in A and E only). Arrows indicate neurons without appreciable NPC1 protein and high filipin staining. Arrowheads indicate neurons successfully transduced by the AAV9-CamKII-NPC1 with subsequent intense NPC1 staining and reduced filipin labelling. ( C,D ) NPC1 intensity of all Layer V neurons measured, plotted against filipin intensity. ( G,H ) NPC1 intensity of all CA3 hippocampal neurons measured, plotted against filipin intensity. Upper left quadrants in (D,H) indicate the percentage of Npc1 -/- neurons corrected to control levels with AAV9-CamKII-NPC1 treatment. Imaged density of AAV-CamKII-GFP ( I,J ) and AAV9-CamKII-NPC1 ( K,L ) incorporation in the Layer V cortex ( I,K ) and CA3 hippocampus ( J,L ), with quantification in ( M,N ). ns = non-significant.

Techniques Used: Transduction, Immunohistochemistry, Imaging, Staining

Effect of AAV9-EF1a-NPC1 vs AAV9-CamKII-NPC1 treatment in brain of Npc1 -/- mice. ( A–E ) Immunohistochemical imaging of cholesterol (visualized with filipin labelling and noted as white intracellular punctae) in the pons, as outlined by box in (A). Npc1 +/+ mice exhibit no cholesterol storage (B) while abundance of cholesterol laden vesicles are apparent in Npc1 -/- mice (C). Npc1 -/- mice treated with AAV9-EF1a-NPC1 exhibit reduced cholesterol accumulation (D) and Npc1 -/- mice treated with AAV9-CamKII-NPC1 show intermediate correction of pathology (E). ( F–I ) Magnification of region outlined by box in (C) for figures (B–E) to facilitate easier visual comparison of cholesterol storage between treatment groups. Scale bars = 100 µm.
Figure Legend Snippet: Effect of AAV9-EF1a-NPC1 vs AAV9-CamKII-NPC1 treatment in brain of Npc1 -/- mice. ( A–E ) Immunohistochemical imaging of cholesterol (visualized with filipin labelling and noted as white intracellular punctae) in the pons, as outlined by box in (A). Npc1 +/+ mice exhibit no cholesterol storage (B) while abundance of cholesterol laden vesicles are apparent in Npc1 -/- mice (C). Npc1 -/- mice treated with AAV9-EF1a-NPC1 exhibit reduced cholesterol accumulation (D) and Npc1 -/- mice treated with AAV9-CamKII-NPC1 show intermediate correction of pathology (E). ( F–I ) Magnification of region outlined by box in (C) for figures (B–E) to facilitate easier visual comparison of cholesterol storage between treatment groups. Scale bars = 100 µm.

Techniques Used: Mouse Assay, Immunohistochemistry, Imaging

10) Product Images from "A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts"

Article Title: A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts

Journal: JIMD Reports

doi: 10.1007/8904_2015_507

Micrograph of Fabry fibroblasts stained with filipin for cholesterol, left , and Gb3, right shows a typical granular staining for lysosomes
Figure Legend Snippet: Micrograph of Fabry fibroblasts stained with filipin for cholesterol, left , and Gb3, right shows a typical granular staining for lysosomes

Techniques Used: Staining

11) Product Images from "Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis"

Article Title: Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015054

Filipin staining of free cholesterol in NPC1 cells treated with HPB-CD for 20 hours. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values from 3 independent experiments. * P
Figure Legend Snippet: Filipin staining of free cholesterol in NPC1 cells treated with HPB-CD for 20 hours. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values from 3 independent experiments. * P

Techniques Used: Staining

12) Product Images from "Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons"

Article Title: Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons

Journal: Nature Communications

doi: 10.1038/ncomms6514

VEGF ameliorates sphingolipid imbalance in NP–C iPSC neurons. ( a ) Left, normal, hNPC-3 and hNPC-17 iPSCs generated nestin-positive neuroprogenitor cells (scale bar, 50 μm). Right, representative images of immunocytochemical staining the β-III tubulin following neural differentiation (scale bar, 50 μm). ( b ) hNPC-3 and hNPC-17 neurons were treated with human BM-MSCs, VEGF tg BM-MSCs or recombinant VEGF (10 ng ml −1 ). Three days after treatment, VEGF levels were measured in cell lysates. ( c – f ) SphK activity ( c ), sphingosine ( d ), S1P ( e ) and sphingomyelin ( f ) were measured in normal iPSC neurons and hNPC neurons with or without treatment. ( g ) Filipin staining of unesterified cholesterol in hNPC-3 neurons with or without treatment of recombinant VEGF for 3 days (scale bar, 50 μm). Quantification of filipin fluorescence intensities normalized to normal neurons. Unesterified cholesterol levels in normal iPSC neurons and hNPC neurons with or without treatment were measured ( n =6 per group). ( h ) Effect of the VEGFR2 inhibitor on VEGF mediated sphingolipid modulation. hNPC-3 neurons were pretreated with PTK787 at 10 μM for 1 day and were treated for 3 days with 10 ng ml −1 VEGF and then assayed for SphK activity, sphingosine and S1P ( n =7 per group). ( i , j ) Effect of NPC1 knockdown on sphingolipid factors in normal iPSC neurons. ( i ) Three days after NPC1 siRNA transfection, we measured the levels of NP C1 mRNA and VEGF expression. ( j ) SphK activity, sphingosine and S1P were measured in normal iPSC neurons treated with control or NPC1 siRNA (control, n =7; NPC1 siRNA, n =9). ( k ) Effect of VEGF knockdown on sphingolipid factors in normal iPSC neurons. Three days after VEGF siRNA transfection, we measured the levels of VEGF mRNA, SphK activity, sphingosine and S1P in normal iPSC neurons ( n =7 per group). ( l ) Effect of a specific SphK1 inhibitor on sphingolipid factors in normal iPSC neurons. Normal neurons were treated with or without 20 μM SK1-I for 6 h. Lipids were extracted and sphingosine, S1P and unesterified cholesterol levels were determined ( n =6 per group). b – h , one-way analysis of variance, Tukey’s post hoc test. i – l , Student’s t -test. * P
Figure Legend Snippet: VEGF ameliorates sphingolipid imbalance in NP–C iPSC neurons. ( a ) Left, normal, hNPC-3 and hNPC-17 iPSCs generated nestin-positive neuroprogenitor cells (scale bar, 50 μm). Right, representative images of immunocytochemical staining the β-III tubulin following neural differentiation (scale bar, 50 μm). ( b ) hNPC-3 and hNPC-17 neurons were treated with human BM-MSCs, VEGF tg BM-MSCs or recombinant VEGF (10 ng ml −1 ). Three days after treatment, VEGF levels were measured in cell lysates. ( c – f ) SphK activity ( c ), sphingosine ( d ), S1P ( e ) and sphingomyelin ( f ) were measured in normal iPSC neurons and hNPC neurons with or without treatment. ( g ) Filipin staining of unesterified cholesterol in hNPC-3 neurons with or without treatment of recombinant VEGF for 3 days (scale bar, 50 μm). Quantification of filipin fluorescence intensities normalized to normal neurons. Unesterified cholesterol levels in normal iPSC neurons and hNPC neurons with or without treatment were measured ( n =6 per group). ( h ) Effect of the VEGFR2 inhibitor on VEGF mediated sphingolipid modulation. hNPC-3 neurons were pretreated with PTK787 at 10 μM for 1 day and were treated for 3 days with 10 ng ml −1 VEGF and then assayed for SphK activity, sphingosine and S1P ( n =7 per group). ( i , j ) Effect of NPC1 knockdown on sphingolipid factors in normal iPSC neurons. ( i ) Three days after NPC1 siRNA transfection, we measured the levels of NP C1 mRNA and VEGF expression. ( j ) SphK activity, sphingosine and S1P were measured in normal iPSC neurons treated with control or NPC1 siRNA (control, n =7; NPC1 siRNA, n =9). ( k ) Effect of VEGF knockdown on sphingolipid factors in normal iPSC neurons. Three days after VEGF siRNA transfection, we measured the levels of VEGF mRNA, SphK activity, sphingosine and S1P in normal iPSC neurons ( n =7 per group). ( l ) Effect of a specific SphK1 inhibitor on sphingolipid factors in normal iPSC neurons. Normal neurons were treated with or without 20 μM SK1-I for 6 h. Lipids were extracted and sphingosine, S1P and unesterified cholesterol levels were determined ( n =6 per group). b – h , one-way analysis of variance, Tukey’s post hoc test. i – l , Student’s t -test. * P

Techniques Used: Generated, Staining, Recombinant, Activity Assay, Fluorescence, Transfection, Expressing

13) Product Images from "β-Cyclodextrin-threaded Biocleavable Polyrotaxanes Ameliorate Impaired Autophagic Flux in Niemann-Pick Type C Disease *"

Article Title: β-Cyclodextrin-threaded Biocleavable Polyrotaxanes Ameliorate Impaired Autophagic Flux in Niemann-Pick Type C Disease *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.636803

The effect of HP-β-CD and a biocleavable polyrotaxanes on the lysosomal cholesterol accumulation and impaired autophagy in NPC1 fibroblasts. A , filipin staining for cholesterol and anti-LC3 staining for autophagosomes in the normal and NPC1 fibroblasts
Figure Legend Snippet: The effect of HP-β-CD and a biocleavable polyrotaxanes on the lysosomal cholesterol accumulation and impaired autophagy in NPC1 fibroblasts. A , filipin staining for cholesterol and anti-LC3 staining for autophagosomes in the normal and NPC1 fibroblasts

Techniques Used: Staining

The effect of α-CD-threaded PRXs and other β-CD derivatives on autophagic flux. A , filipin staining for cholesterol in normal and NPC1 fibroblasts treated with α-CD (10 m m ), PEG/α-CD PRX (1 m m α-CD), and P123/α-CD
Figure Legend Snippet: The effect of α-CD-threaded PRXs and other β-CD derivatives on autophagic flux. A , filipin staining for cholesterol in normal and NPC1 fibroblasts treated with α-CD (10 m m ), PEG/α-CD PRX (1 m m α-CD), and P123/α-CD

Techniques Used: Staining

14) Product Images from "Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)"

Article Title: Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

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

doi: 10.1073/pnas.091090798

Labeling of A431 cells with BCθ or filipin. Monolayer A431 cells were incubated with serum-free DMEM for 2 h, fixed, and then incubated with either BCθ or filipin. BCθ-treated cells were then incubated with cy3-avidin. ( a – d ) Binding of BCθ ( a and b ) or filipin ( c and d ) to 2OHpβCD-treated cells ( b and d ) or untreated controls ( a and c ). Depletion of cholesterol completely abolished BCθ binding, whereas filipin binding was retained significantly. ( Left ) Phase contrast; ( Right ) fluorescence staining.
Figure Legend Snippet: Labeling of A431 cells with BCθ or filipin. Monolayer A431 cells were incubated with serum-free DMEM for 2 h, fixed, and then incubated with either BCθ or filipin. BCθ-treated cells were then incubated with cy3-avidin. ( a – d ) Binding of BCθ ( a and b ) or filipin ( c and d ) to 2OHpβCD-treated cells ( b and d ) or untreated controls ( a and c ). Depletion of cholesterol completely abolished BCθ binding, whereas filipin binding was retained significantly. ( Left ) Phase contrast; ( Right ) fluorescence staining.

Techniques Used: Labeling, Incubation, Avidin-Biotin Assay, Binding Assay, Fluorescence, Staining

15) Product Images from "Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling D⃞"

Article Title: Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-05-0432

Rab4-containing endosomes in NPFs are enriched in cholesterol, as assessed by filipin staining. Normal HSFs or NPFs either were pulse-labeled with FITC-Tfn for 1 h at 16°C to label the EEs, washed, fixed, and stained with filipin to detect cholesterol,
Figure Legend Snippet: Rab4-containing endosomes in NPFs are enriched in cholesterol, as assessed by filipin staining. Normal HSFs or NPFs either were pulse-labeled with FITC-Tfn for 1 h at 16°C to label the EEs, washed, fixed, and stained with filipin to detect cholesterol,

Techniques Used: Staining, Labeling

16) Product Images from ""

Article Title:

Journal:

doi: 10.1091/mbc.E06-10-0924

Cholesterol increases when CHO cells reach confluence. Subconfluent and confluent cells were doubly labeled with filipin and anti-γ-tubulin. Bar, 10 μm. Images were obtained under the same acquisition conditions (exposure time 1.97 s with
Figure Legend Snippet: Cholesterol increases when CHO cells reach confluence. Subconfluent and confluent cells were doubly labeled with filipin and anti-γ-tubulin. Bar, 10 μm. Images were obtained under the same acquisition conditions (exposure time 1.97 s with

Techniques Used: Labeling

17) Product Images from "Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease"

Article Title: Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease

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

doi: 10.1073/pnas.081070898

Late endocytic trafficking is blocked by cholesterol accumulation in CT60 CHO cells expressing SSD-NPC1-GFP. ( A ) Western blot analysis with anti-GFP antibody of total solubilized protein from SSD-NPC1-GFP-expressing WT CHO cells ( Left ) and EGFP-expressing WT CHO cells ( Right ). The predicted molecular mass of GFP (27 kDa) combined with glycosylated SSD-NPC1 (170 kDa) is comparable to the 200-kDa band ( Left ). ( B–E ) CT 60 CHO cells expressing SSD-NPC1-GFP fail to clear cholesterol. Seventy-two hours after transfection, cells were fixed, stained, and labeled with filipin ( B , blue) to visualize cholesterol by confocal microscopy. SSD-NPC1-GFP ( C , green) was also detected by indirect immunofluorescence ( D , red). ( E ) Cholesterol-laden lysosomes in an SSD-NPC1-GFP transfected CT60 CHO cell are labeled by endocytic uptake of rhodamine-dextran. In this merged image, SSD-NPC1-GFP (green) is present as rings at the periphery of rhodamine-dextran (red) in the cholesterol-laden core of lysosomes. B–E are confocal images. [Bar = 10 μm ( B–D ) and 2 μm ( E ).]
Figure Legend Snippet: Late endocytic trafficking is blocked by cholesterol accumulation in CT60 CHO cells expressing SSD-NPC1-GFP. ( A ) Western blot analysis with anti-GFP antibody of total solubilized protein from SSD-NPC1-GFP-expressing WT CHO cells ( Left ) and EGFP-expressing WT CHO cells ( Right ). The predicted molecular mass of GFP (27 kDa) combined with glycosylated SSD-NPC1 (170 kDa) is comparable to the 200-kDa band ( Left ). ( B–E ) CT 60 CHO cells expressing SSD-NPC1-GFP fail to clear cholesterol. Seventy-two hours after transfection, cells were fixed, stained, and labeled with filipin ( B , blue) to visualize cholesterol by confocal microscopy. SSD-NPC1-GFP ( C , green) was also detected by indirect immunofluorescence ( D , red). ( E ) Cholesterol-laden lysosomes in an SSD-NPC1-GFP transfected CT60 CHO cell are labeled by endocytic uptake of rhodamine-dextran. In this merged image, SSD-NPC1-GFP (green) is present as rings at the periphery of rhodamine-dextran (red) in the cholesterol-laden core of lysosomes. B–E are confocal images. [Bar = 10 μm ( B–D ) and 2 μm ( E ).]

Techniques Used: Expressing, Western Blot, Transfection, Staining, Labeling, Confocal Microscopy, Immunofluorescence

Expression, functional analysis, and subcellular localization of NPC1-GFP in CT60 CHO cells. ( A ) CT60 CHO cell transfected with NPC1-GFP (green) was ( B ) immunostained with anti-human NPC1 antiserum (red) and ( C ) stained with filipin (blue) to visualize cholesterol-loaded lysosomes. ( D ) Western blot analysis with anti-GFP antibody of total solubilized protein from NPC1-GFP-expressing ( Left ) and EGFP-expressing ( Right ) WT CHO cells. The predicted molecular mass of GFP (27 kDa) combined with glycosylated NPC1 (170 kDa and 190 kDa) is comparable to the 200- and 220-kDa bands. Identical results were obtained with total soluble protein from NPC1-GFP transfected CT60 CHO cells (data not shown). ( E ) Merged image of CT60 CHO cell transfected with NPC1-GFP (green) and immunostained with LAMP2 (red). Colocalization is represented by yellow. ( F ) Merged image of a NPC1-GFP-expressing WT CHO cell. Cells were transfected with NPC1-GFP (green) and pulsed with Texas Red–transferrin (red) for 10 min. A–C, E , and F are confocal images. (Bar = 10 μm.)
Figure Legend Snippet: Expression, functional analysis, and subcellular localization of NPC1-GFP in CT60 CHO cells. ( A ) CT60 CHO cell transfected with NPC1-GFP (green) was ( B ) immunostained with anti-human NPC1 antiserum (red) and ( C ) stained with filipin (blue) to visualize cholesterol-loaded lysosomes. ( D ) Western blot analysis with anti-GFP antibody of total solubilized protein from NPC1-GFP-expressing ( Left ) and EGFP-expressing ( Right ) WT CHO cells. The predicted molecular mass of GFP (27 kDa) combined with glycosylated NPC1 (170 kDa and 190 kDa) is comparable to the 200- and 220-kDa bands. Identical results were obtained with total soluble protein from NPC1-GFP transfected CT60 CHO cells (data not shown). ( E ) Merged image of CT60 CHO cell transfected with NPC1-GFP (green) and immunostained with LAMP2 (red). Colocalization is represented by yellow. ( F ) Merged image of a NPC1-GFP-expressing WT CHO cell. Cells were transfected with NPC1-GFP (green) and pulsed with Texas Red–transferrin (red) for 10 min. A–C, E , and F are confocal images. (Bar = 10 μm.)

Techniques Used: Expressing, Functional Assay, Transfection, Staining, Western Blot

18) Product Images from "Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks"

Article Title: Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks

Journal: Orphanet Journal of Rare Diseases

doi: 10.1186/1750-1172-8-144

Cholesterol accumulation in fibroblasts, hiPSCs, and neural progenitor cells. Cholesterol accumulation is one of the hallmarks of NPC1 disease. Filipin stainings of fibroblast ( A,B , shown in blue) are used for diagnostics, where fibroblasts of NPC1 patients with a “classic” biochemical phenotype demonstrate a clear perinuclear accumulation (A) in contrast to fibroblasts of an unaffected individual (B) . These differences were found in hiPSCs (C,D) and neural progenitor cells (hNPCs, E,F ) derived from mutNPC1 and wtNPC1 fibroblasts. (scale bar = 100 μm). A quantification of the amount of cholesterol (G) in fibroblasts, iPSCs, and hNPCs revealed elevated cholesterol levels in mutNPC1 cell lines (black bars) in contrast to wtNPC1 cell lines (white bars). The total amount differed slightly between the cell lines but the relative proportion was comparable.
Figure Legend Snippet: Cholesterol accumulation in fibroblasts, hiPSCs, and neural progenitor cells. Cholesterol accumulation is one of the hallmarks of NPC1 disease. Filipin stainings of fibroblast ( A,B , shown in blue) are used for diagnostics, where fibroblasts of NPC1 patients with a “classic” biochemical phenotype demonstrate a clear perinuclear accumulation (A) in contrast to fibroblasts of an unaffected individual (B) . These differences were found in hiPSCs (C,D) and neural progenitor cells (hNPCs, E,F ) derived from mutNPC1 and wtNPC1 fibroblasts. (scale bar = 100 μm). A quantification of the amount of cholesterol (G) in fibroblasts, iPSCs, and hNPCs revealed elevated cholesterol levels in mutNPC1 cell lines (black bars) in contrast to wtNPC1 cell lines (white bars). The total amount differed slightly between the cell lines but the relative proportion was comparable.

Techniques Used: Derivative Assay

19) Product Images from "Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution [S]"

Article Title: Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.D018184

Filipin labeling of cells treated with hit compounds. HeLa cells were treated without or with 12.5 μg/ml hit compounds in 10% LPDS-containing medium for 18 h. The cells were labeled with filipin and anti GM130 antibodies or anti CD63 antibodies, as described in MATERIALS AND METHODS. Images were obtained under a Zeiss LSM510 confocal microscope with 63× objective. Bar = 20 μm.
Figure Legend Snippet: Filipin labeling of cells treated with hit compounds. HeLa cells were treated without or with 12.5 μg/ml hit compounds in 10% LPDS-containing medium for 18 h. The cells were labeled with filipin and anti GM130 antibodies or anti CD63 antibodies, as described in MATERIALS AND METHODS. Images were obtained under a Zeiss LSM510 confocal microscope with 63× objective. Bar = 20 μm.

Techniques Used: Labeling, Microscopy

EGFP-D4 and filipin labeling of HeLa cells treated with lovastatin and U18666A. A: HeLa cells were treated with or without 8 μM lovastatin and 100 μM mevalonate, or 2 μg/ml U18666A in FBS- or LPDS-containing medium for 18 h. Cells were labeled with EGFP-D4 and Hoechst 33342 as described in MATERIALS AND METHODS. Images were acquired using In Cell Analyzer 1000 with a 20× objective. Bar = 20 μm. B: Fluorescence intensity of EGFP-D4 bound to cells was normalized by cell number as described in MATERIALS AND METHODS. Data represent the mean ± SD (n = 4). C: HeLa cells were treated with 8 μM lovastatin and 100 μM mevalonate, or 2 μg/ml U18666A in LPDS-containing medium for 18 h. The cells were labeled with filipin as described in MATERIALS AND METHODS. Images were acquired using In Cell Analyzer 1000 with a 20× objective. Bar = 20 μm.
Figure Legend Snippet: EGFP-D4 and filipin labeling of HeLa cells treated with lovastatin and U18666A. A: HeLa cells were treated with or without 8 μM lovastatin and 100 μM mevalonate, or 2 μg/ml U18666A in FBS- or LPDS-containing medium for 18 h. Cells were labeled with EGFP-D4 and Hoechst 33342 as described in MATERIALS AND METHODS. Images were acquired using In Cell Analyzer 1000 with a 20× objective. Bar = 20 μm. B: Fluorescence intensity of EGFP-D4 bound to cells was normalized by cell number as described in MATERIALS AND METHODS. Data represent the mean ± SD (n = 4). C: HeLa cells were treated with 8 μM lovastatin and 100 μM mevalonate, or 2 μg/ml U18666A in LPDS-containing medium for 18 h. The cells were labeled with filipin as described in MATERIALS AND METHODS. Images were acquired using In Cell Analyzer 1000 with a 20× objective. Bar = 20 μm.

Techniques Used: Labeling, Fluorescence

Binding of EGFP-D4 and filipin to cholesterol-containing liposomes. A: EGFP-D4 was incubated with PC/cholesterol or SM/cholesterol liposomes (MLVs) for 30 min at room temperature. After centrifugation, the pellet fractions were subjected to SDS-PAGE followed by CBB staining. B: EGFP-D4 bound to PC/cholesterol (filled bar) and SM/cholesterol liposomes (open bar) was stained with SYPRO Ruby, and quantitated as described under MATERIALS AND METHODS. Data are the mean ± average deviation of two independent experiments. C: Filipin (10 μM) was incubated with 100 μM liposomes (SUVs) for 30 min at room temperature. Absorbance at 320 nm (Peak 3) and 356 nm (Peak 1) was determined as described under MATERIALS AND METHODS. Data represent the mean ± SD (n = 3).
Figure Legend Snippet: Binding of EGFP-D4 and filipin to cholesterol-containing liposomes. A: EGFP-D4 was incubated with PC/cholesterol or SM/cholesterol liposomes (MLVs) for 30 min at room temperature. After centrifugation, the pellet fractions were subjected to SDS-PAGE followed by CBB staining. B: EGFP-D4 bound to PC/cholesterol (filled bar) and SM/cholesterol liposomes (open bar) was stained with SYPRO Ruby, and quantitated as described under MATERIALS AND METHODS. Data are the mean ± average deviation of two independent experiments. C: Filipin (10 μM) was incubated with 100 μM liposomes (SUVs) for 30 min at room temperature. Absorbance at 320 nm (Peak 3) and 356 nm (Peak 1) was determined as described under MATERIALS AND METHODS. Data represent the mean ± SD (n = 3).

Techniques Used: Binding Assay, Incubation, Centrifugation, SDS Page, Staining

Related Articles

Transfection:

Article Title: Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization
Article Snippet: Transiently transfected cells were fixed with 3% paraformaldehyde in PBS for 30 min. .. Cells were washed in PBS (three times for 5 min), quenched with 1.5 mg/ml glycine in PBS for 10 min, and stained with 0.05 mg/ml of filipin (Polysciences) in PBS for 30 min. After washing in PBS (three times for 5 min), slides were mounted with coverslips in phenylenediamine/glycerol.

Article Title: Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis
Article Snippet: Filipin was from Polysciences Inc., (Warrington, PA). .. FuGENE™ 6 transfection reagent was from Roche Diagnostics.

Article Title: PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport
Article Snippet: Filipin was from Polysciences, Inc. (Warrington, PA). .. Lumilight Plus substrate and FuGENETM 6 transfection reagent were both from Roche Diagnostics (Indianapolis, IN).

Fluorescence:

Article Title: Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization
Article Snippet: Cells were washed in PBS (three times for 5 min), quenched with 1.5 mg/ml glycine in PBS for 10 min, and stained with 0.05 mg/ml of filipin (Polysciences) in PBS for 30 min. After washing in PBS (three times for 5 min), slides were mounted with coverslips in phenylenediamine/glycerol. .. Intense filipin fluorescence staining of cholesterol in large perinuclear granules is characteristic of the lysosomal cholesterol accumulation in NPC cells ( – , , ).

Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds
Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature. .. Images were taken using a Zeiss Observer Z1 inverted compound fluorescence microscope with a Calibri.2 LED lighting system and a CCD c amera (Carl Zeiss).

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
Article Snippet: Paragraph title: Fluorescence microscopy ... After rinses with 0.17 M potassium dihydrogen phosphate and 30 mM sodium citrate (pH 5.8), cells were treated for 5 min with 0.025% Nonidet P-40 in PBS, then with 1 mg/ml sodium borohydride in Tris-buffered saline (pH 8.2), and were labeled for 10 min in 10 μg/ml filipin (Polysciences, Warminster, PA, USA) in PBS.

Synthesized:

Article Title: Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells
Article Snippet: BODIPY-LacCer was synthesized as described ( ). .. Filipin was purchased from Polysciences Inc. (Warrington, Pennsylvania, USA), Nile Red from Eastman Kodak (Rochester, New York, USA), and rhodamine-labeled cholera toxin B subunit (Rh-CtxB) from List Biological Laboratories Inc. (Campbell, California, USA).

Immunohistochemistry:

Article Title: Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1
Article Snippet: Paragraph title: Immunohistochemistry ... Filipin (Polysciences Inc.) staining was performed at a final concentration of 50 µg/ml in PBSt.

Microscopy:

Article Title: A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts
Article Snippet: Filipin was purchased from Polysciences, Warrington, PA; mouse monoclonal anti-Gb3 antibody was from Seikagaku, Falmouth, MA; and Alexa-568 tagged IgG goat anti-mouse antibody and DiI-LDL were from Life Technologies, Grand Island, NY. .. Glass and plastic microscope culture chamber slides were from Nunc, Inc., Naperville, IL.

Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds
Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature. .. Images were taken using a Zeiss Observer Z1 inverted compound fluorescence microscope with a Calibri.2 LED lighting system and a CCD c amera (Carl Zeiss).

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
Article Snippet: Paragraph title: Fluorescence microscopy ... After rinses with 0.17 M potassium dihydrogen phosphate and 30 mM sodium citrate (pH 5.8), cells were treated for 5 min with 0.025% Nonidet P-40 in PBS, then with 1 mg/ml sodium borohydride in Tris-buffered saline (pH 8.2), and were labeled for 10 min in 10 μg/ml filipin (Polysciences, Warminster, PA, USA) in PBS.

Article Title: An "Exacerbate-reverse" Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease *
Article Snippet: Cholesterol and the glycosphingolipid globotriosylceramide (GL-3) accumulation were monitored using filipin (Polysciences) or verotoxin, respectively, as described previously ( , ). .. Images were obtained sequentially using confocal laser microscopy, and quantification analysis was performed using ImageJ64 or MetaVue ( ).

Mouse Assay:

Article Title: Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1
Article Snippet: Mice were euthanized by CO2 asphyxiation and transcardially perfused with 4% paraformaldehyde in phosphate buffer. .. Filipin (Polysciences Inc.) staining was performed at a final concentration of 50 µg/ml in PBSt.

Concentration Assay:

Article Title: Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1
Article Snippet: .. Filipin (Polysciences Inc.) staining was performed at a final concentration of 50 µg/ml in PBSt. ..

Incubation:

Article Title: Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons
Article Snippet: .. Filipin staining Cells and cerebellar sections were fixed with 4% paraformaldehyde for 15 min, washed with PBS and incubated for 30 min with 100 μg ml−1 filipin (Polysciences) in PBS. ..

Article Title: Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease
Article Snippet: After incubation for 24 h, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. .. The cells were stained with filipin (PolySciences, Warrington, PA, USA) (50 μg/mL) for 45 min at room temperature.

Article Title: Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1
Article Snippet: The sections were incubated overnight at 4 °C with either rabbit anti-calbindin (1:3000, Swant), rabbit anti-NPC1 ( ) (1:2000, generous gift of Dr. Daniel Ory, Washington University in St. Louis), mouse anti-NeuN (1:1000, Millipore) in PBSt, or rat anti-CD68 (1:1000, AbD Serotec) and the primaries detected using DyLight-488 goat anti-rabbit/mouse IgG or Alexa-594 anti-rabbit (1:1000 in PBSt, Vector Labs) or Alexa-488 anti-rat (1:300, ThermoFisher Scientific). .. Filipin (Polysciences Inc.) staining was performed at a final concentration of 50 µg/ml in PBSt.

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
Article Snippet: Fluorescence microscopy For labeling of LDs, yeast cells were incubated with 0.5 μg/ml BODIPY493/503 (Thermo Fisher) for 10 min, and rinsed with 50 mM Tris-HCl (pH 7.5) immediately before microscopy ( ). .. After rinses with 0.17 M potassium dihydrogen phosphate and 30 mM sodium citrate (pH 5.8), cells were treated for 5 min with 0.025% Nonidet P-40 in PBS, then with 1 mg/ml sodium borohydride in Tris-buffered saline (pH 8.2), and were labeled for 10 min in 10 μg/ml filipin (Polysciences, Warminster, PA, USA) in PBS.

Article Title: An "Exacerbate-reverse" Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease *
Article Snippet: Cholesterol and the glycosphingolipid globotriosylceramide (GL-3) accumulation were monitored using filipin (Polysciences) or verotoxin, respectively, as described previously ( , ). .. The trafficking of lactosylceramide was monitored using BODIPY-LacCer; cells in a glass bottom dish (Iwaki, Japan) were washed with Hanks' buffered saline solution, incubated with 5 μ m BODIPY-LacCer with BSA complex (Molecular Probes) for 30 min at 4 °C, and incubated with DMEM 10% FBS at 37 °C for 30 min as described in the manufacturer's protocol.

Confocal Laser Scanning Microscopy:

Article Title: Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease
Article Snippet: The cells were stained with filipin (PolySciences, Warrington, PA, USA) (50 μg/mL) for 45 min at room temperature. .. The confocal laser scanning microscopic (CLSM) observations were performed on a FluoView FV10i (Olympus, Tokyo, Japan) equipped with a 60× water-immersion objective lens (N/A 1.2) and a diode laser.

Activity Assay:

Article Title: A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts
Article Snippet: Fabry fibroblasts GM 00107 with 0.0% of normal control alpha-galactosidase-A enzyme activity were from the Coriell Institute of Medical Research (Camden NJ, USA). .. Filipin was purchased from Polysciences, Warrington, PA; mouse monoclonal anti-Gb3 antibody was from Seikagaku, Falmouth, MA; and Alexa-568 tagged IgG goat anti-mouse antibody and DiI-LDL were from Life Technologies, Grand Island, NY.

Labeling:

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
Article Snippet: .. After rinses with 0.17 M potassium dihydrogen phosphate and 30 mM sodium citrate (pH 5.8), cells were treated for 5 min with 0.025% Nonidet P-40 in PBS, then with 1 mg/ml sodium borohydride in Tris-buffered saline (pH 8.2), and were labeled for 10 min in 10 μg/ml filipin (Polysciences, Warminster, PA, USA) in PBS. .. In experiments that looked at filipin staining and Vph1–GFP simultaneously, cells were fixed for 1 hr with 0.25% glutaraldehyde and 5% formaldehyde in 100 mM sodium phosphate buffer (pH 7.4).

Expressing:

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
Article Snippet: After rinses with 0.17 M potassium dihydrogen phosphate and 30 mM sodium citrate (pH 5.8), cells were treated for 5 min with 0.025% Nonidet P-40 in PBS, then with 1 mg/ml sodium borohydride in Tris-buffered saline (pH 8.2), and were labeled for 10 min in 10 μg/ml filipin (Polysciences, Warminster, PA, USA) in PBS. .. The stained yeast cells, as well as those expressing GFP- or mRFP-tagged proteins, were mounted on a glass slide and observed under an AxioImager or Axiovert microscope using a 63× NA1.4 Apochromat objective lens (Zeiss, Oberkochen, Germany).

Modification:

Article Title: PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport
Article Snippet: Materials Dulbecco’s Modified Eagle Medium (DMEM), trypsin, L-glutamine, gentamicin, and NuPage gels and buffers were obtained from Invitrogen (Carlsbad, CA) while FBS was from Hyclone, Thermo Scientific (Rockford, IL). .. Filipin was from Polysciences, Inc. (Warrington, PA).

Staining:

Article Title: Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons
Article Snippet: .. Filipin staining Cells and cerebellar sections were fixed with 4% paraformaldehyde for 15 min, washed with PBS and incubated for 30 min with 100 μg ml−1 filipin (Polysciences) in PBS. ..

Article Title: Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization
Article Snippet: .. Cells were washed in PBS (three times for 5 min), quenched with 1.5 mg/ml glycine in PBS for 10 min, and stained with 0.05 mg/ml of filipin (Polysciences) in PBS for 30 min. After washing in PBS (three times for 5 min), slides were mounted with coverslips in phenylenediamine/glycerol. ..

Article Title: Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease
Article Snippet: .. The cells were stained with filipin (PolySciences, Warrington, PA, USA) (50 μg/mL) for 45 min at room temperature. .. The confocal laser scanning microscopic (CLSM) observations were performed on a FluoView FV10i (Olympus, Tokyo, Japan) equipped with a 60× water-immersion objective lens (N/A 1.2) and a diode laser.

Article Title: Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1
Article Snippet: .. Filipin (Polysciences Inc.) staining was performed at a final concentration of 50 µg/ml in PBSt. ..

Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds
Article Snippet: .. After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature. .. Stained embryos/larvae were rinsed and stored in 1× PBST before imaging.

Article Title: Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
Article Snippet: For sterol staining, yeast cells were fixed for 30 min with 1% glutaraldehyde in 40 mM potassium phosphate buffer (pH 7) and 0.5 mM magnesium choloride, and treated for 10 min with 0.7% 2-mercaptoethanol in 0.2 M Tris and 20 mM EDTA. .. After rinses with 0.17 M potassium dihydrogen phosphate and 30 mM sodium citrate (pH 5.8), cells were treated for 5 min with 0.025% Nonidet P-40 in PBS, then with 1 mg/ml sodium borohydride in Tris-buffered saline (pH 8.2), and were labeled for 10 min in 10 μg/ml filipin (Polysciences, Warminster, PA, USA) in PBS.

Imaging:

Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds
Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature. .. Stained embryos/larvae were rinsed and stored in 1× PBST before imaging.

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    Polysciences inc filipin
    Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by <t>filipin</t> fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P
    Filipin, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by filipin fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P

    Journal: PLoS ONE

    Article Title: PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport

    doi: 10.1371/journal.pone.0074169

    Figure Lengend Snippet: Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by filipin fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P

    Article Snippet: Filipin was from Polysciences, Inc. (Warrington, PA).

    Techniques: Activation Assay, Fluorescence

    Effects of fatty acids on vimentin solubilization and the NPC1 phenotype. (A) Human NPC1 3123 cells were treated with 50µg/ml linoleic or oleic acid for 24 hrs, after which the levels of soluble vimentin were analyzed by Western blotting. Cholesterol storage in NPC1 CHO (B) or human 3123 (C) cells was analyzed by filipin staining. Fluorescence intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. * and *** denote statistically significant differences between treated and untreated cells with P

    Journal: PLoS ONE

    Article Title: PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport

    doi: 10.1371/journal.pone.0074169

    Figure Lengend Snippet: Effects of fatty acids on vimentin solubilization and the NPC1 phenotype. (A) Human NPC1 3123 cells were treated with 50µg/ml linoleic or oleic acid for 24 hrs, after which the levels of soluble vimentin were analyzed by Western blotting. Cholesterol storage in NPC1 CHO (B) or human 3123 (C) cells was analyzed by filipin staining. Fluorescence intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. * and *** denote statistically significant differences between treated and untreated cells with P

    Article Snippet: Filipin was from Polysciences, Inc. (Warrington, PA).

    Techniques: Western Blot, Staining, Fluorescence

    Effects of transient PKC expression on the NPC1 phenotype. M12 cells were transfected with PKC isoforms or Rab9 for 48 hrs and then analyzed by filipin staining for cholesterol storage. Cells positive for transfection stain positive for GFP (left panel) and show decreased filipin staining (outlined cells, right panel) compared to surrounding untransfected cells, confirming the role of PKCs in mobilizing stored cholesterol from the NPC1 endosomes. Bar, 20μ.

    Journal: PLoS ONE

    Article Title: PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport

    doi: 10.1371/journal.pone.0074169

    Figure Lengend Snippet: Effects of transient PKC expression on the NPC1 phenotype. M12 cells were transfected with PKC isoforms or Rab9 for 48 hrs and then analyzed by filipin staining for cholesterol storage. Cells positive for transfection stain positive for GFP (left panel) and show decreased filipin staining (outlined cells, right panel) compared to surrounding untransfected cells, confirming the role of PKCs in mobilizing stored cholesterol from the NPC1 endosomes. Bar, 20μ.

    Article Snippet: Filipin was from Polysciences, Inc. (Warrington, PA).

    Techniques: Expressing, Transfection, Staining

    Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: Unesterified cholesterol accumulation. (A) Control and mutant larvae were stained with filipin to visualize unesterified cholesterol. Filipin-positive puncta were initially observed in mutant larvae at 2 dpf in the area of the yolk sac extension and became more numerous with a wider distribution in older larvae. Scale bars: 200 µm. (B) Embryos were injected with TopFluor-cholesterol at the 1-cell stage and imaged at 7 dpf. Puncta of accumulated cholesterol were observed in the npc1 m/m larvae. Scale bar: 1 mm.

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Mutagenesis, Staining, Injection

    2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: 2HPβCD treatment reduces LysoTracker Red and filipin staining in npc1 m/m neuromasts. (A) Control and mutant larvae can be differentiated at 3 dpf by intense LysoTracker Red (Lyso R) staining of the olfactory placode (arrowheads) at 3 dpf. Scale bars: 100 μm. (B) Mutant larvae selected by Lyso-R-positive olfactory placode staining were treated for 3 days with vehicle (ddH 2 O) or 2.5 mM 2HPβCD starting at 3 dpf. Neuromast Lyso R staining was markedly decreased in 2HPβCD-treated npc1 m/m larvae. Scale bars: 1 mm. (C) Higher magnification view of Lyso R staining, corresponding to the boxed area in B. Scale bar: 100 μm. (D) Lyso R relative intensity was significantly reduced in npc1 m/m larvae treated with 2.5 mM 2HPβCD at 6 dpf. **** P

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Staining, Mutagenesis

    Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

    Journal: Disease Models & Mechanisms

    Article Title: Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

    doi: 10.1242/dmm.034165

    Figure Lengend Snippet: Yolk syncytial layer accumulation of unesterified cholesterol. Plasmids expressing either EGFP or npc1 were injected into the yolk syncytial layer (YSL) of 3.5 hpf embryos. Filipin staining of 2 dpf embryos demonstrated reversal of unesterified cholesterol accumulation in the YSL of mutant larvae expressing npc1 . Scale bar: 200 μm.

    Article Snippet: After extensive rinsing with 1× PBS, fixed embryos/larvae were then stained with filipin staining solution containing 0.5 mg/ml filipin (08707, Polysciences, Warrington, PA, USA) and 1% goat serum in 1× PBS for 2.5 h at room temperature.

    Techniques: Expressing, Injection, Staining, Mutagenesis

    Global histone deacetylase inhibition corrects cholesterol homeostasis in NP-C fibroblasts. Mutant fibroblasts (NPC-26 and NPC-J4) were incubated for 18 h in the presence of 5 μ m SAHA, stained with filipin, and assessed for esterification of LDL-cholesterol.

    Journal: The Journal of Biological Chemistry

    Article Title: An "Exacerbate-reverse" Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease *

    doi: 10.1074/jbc.M111.227645

    Figure Lengend Snippet: Global histone deacetylase inhibition corrects cholesterol homeostasis in NP-C fibroblasts. Mutant fibroblasts (NPC-26 and NPC-J4) were incubated for 18 h in the presence of 5 μ m SAHA, stained with filipin, and assessed for esterification of LDL-cholesterol.

    Article Snippet: Cholesterol and the glycosphingolipid globotriosylceramide (GL-3) accumulation were monitored using filipin (Polysciences) or verotoxin, respectively, as described previously ( , ).

    Techniques: Histone Deacetylase Assay, Inhibition, Mutagenesis, Incubation, Staining

    Lysosomal cholesterol reduction ability of biocleavable HE-SS-PRX against NPC1 cells. (A) Filipin staining for unesterified cholesterol and LysoTracker staining for endosome/lysosome in normal and NPC1 cells (bar: 20 μm). The NPC1 cells were incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at the β-CD concentration of 0.1 mM for 24 h. (B) The amount of intracellular total cholesterol in NPC1 cells incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at various β-CD concentrations for 24 h. The dashed lines represent the amount of intracellular total cholesterol in non-treated normal and NPC1 cells. (C) Time-course of the amount of intracellular total cholesterol in NPC1 cells after incubation with HP-β-CD (β-CD: 10 mM), DM-β-CD (β-CD: 1 mM), non-cleavable HE-PRX (β-CD: 10 mM), and cleavable HE-SS-PRX (β-CD: 0.2 mM) for 24 h, followed by incubation without treatment for the indicated time periods. The closed circles represent the non-treated NPC1 cells. Data are expressed as the mean ± S.D. (n = 3) (* p

    Journal: Scientific Reports

    Article Title: Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease

    doi: 10.1038/srep04356

    Figure Lengend Snippet: Lysosomal cholesterol reduction ability of biocleavable HE-SS-PRX against NPC1 cells. (A) Filipin staining for unesterified cholesterol and LysoTracker staining for endosome/lysosome in normal and NPC1 cells (bar: 20 μm). The NPC1 cells were incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at the β-CD concentration of 0.1 mM for 24 h. (B) The amount of intracellular total cholesterol in NPC1 cells incubated with HP-β-CD, DM-β-CD, non-cleavable HE-PRX, and cleavable HE-SS-PRX at various β-CD concentrations for 24 h. The dashed lines represent the amount of intracellular total cholesterol in non-treated normal and NPC1 cells. (C) Time-course of the amount of intracellular total cholesterol in NPC1 cells after incubation with HP-β-CD (β-CD: 10 mM), DM-β-CD (β-CD: 1 mM), non-cleavable HE-PRX (β-CD: 10 mM), and cleavable HE-SS-PRX (β-CD: 0.2 mM) for 24 h, followed by incubation without treatment for the indicated time periods. The closed circles represent the non-treated NPC1 cells. Data are expressed as the mean ± S.D. (n = 3) (* p

    Article Snippet: The cells were stained with filipin (PolySciences, Warrington, PA, USA) (50 μg/mL) for 45 min at room temperature.

    Techniques: Staining, Incubation, Concentration Assay