filipin Millipore Search Results


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  • 95
    Millipore filipin complex
    The cofilin and actin mutants facilitate the enrichment of sterols at the sites of tombusvirus replication in yeast. (A) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA. Fluorescent microscopic images of yeast cells stained with <t>filipin</t> dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free wt yeast cells, while act1-121 ts yeast and act1-132 ts yeast show uneven distribution of ergosterols in the absence of viral components (right images). (B) Similar experiments as in Panel A, except performed at the semi-permissive temperature (27°C) in act1-132 ts and WT yeasts. (C) Similar experiments as in Panel A, except performed at the semi-permissive temperature (32°C) in act1-121 ts and WT yeasts. (D) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the permissive temperature. Fluorescent microscopic images of yeast cells stained with filipin dye. (E) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the semi-permissive temperature. See further details in panel A. Each experiment was repeated at least three times.
    Filipin Complex, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore filipin
    Electron microscopical visualization of <t>filipin</t> labeled membrane cholesterol. Arrowheads in control (A) and double-deficient fibroblasts (B) indicate the filipin labeling. In control cells labeling was detected in the limiting membranes of small vesicles
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore prepared filipin
    GP73 regulates the transcriptional activity of SREBPs and lipogenesis. ( a ) Immunoblotting analysis of SREBPs activation in HepG2 cells transfected with Flag-GP73 at the indicated doses. α-Tubulin was used as equal loading control. ( b , c ) SREBP-1 promoter activity in HepG2 ( b ) or HL7702 ( c ) cells transfected with Flag-vector or Flag-GP73 under conditions of sterol depletion or repletion. The luciferase activity was measured 36 hrs post transfection. The value was normalized with the corresponding transfection efficiency. ( d , f , h ) QRT-PCR analysis of HMGR ( d ), FASN2 ( f ), and ACC1 ( h ) mRNA abundance in HepG2 cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( e , g , i ) QRT-PCR analysis of HMGR ( e ), FASN2 ( g ), and ACC1 ( i ) mRNA abundance in 293T cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( j ) Fluorescence microscopy of <t>Filipin</t> staining in HepG2 cells transfected with Flag-GP73. Cells were collected at indicated hrs post transfection. ( k , l ) Amplex Red cholesterol assay of cellular cholesterol concentrations in HepG2 ( k ) or HL7702 ( l ) cells transfected with Flag-vector or Flag-GP73. Cells were collected at indicated hrs post transfection. Values were normalized to total cell proteins from control cells transfected with Flag-vector. Cell-based studies were performed at least <t>three</t> independent times with comparable results. Data represent mean ± SEM. Student’s t test was used for statistical analysis: **p
    Prepared Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Millipore filipin dye
    Fluorescence imaging confirms differences in MCA smooth muscle <t>CLR</t> levels among experimental groups. A. Original snapshots showing vascular smooth muscle CLR levels via fluorescence staining of arteries with CLR-sensitive dye <t>filipin.</t> Arteries were collected from rats on high CLR diet + placebo, and high CLR diet + atorvastatin. Several arteries from rats on high CLR diet + atorvastatin were subjected to in vitro CLR enrichment. Artery staining from all experimental groups was performed in parallel to minimize the experimental error and justify the direct comparison of the fluorescence signal intensity. Snapshots on the right show smooth muscle layer in visible light. These images were superposed with fluorescence ones to define the individual myocyte plasma membranes for filipin intensity quantification. In the right top panel, arrows point at the example of individual myocyte’s silhouette (light grey) within vasculature layer. B. Averaged data showing filipin fluorescence signal from high CLR diet + placebo (n = 7), high CLR diet + atorvastatin (n = 10), and high CLR diet + atorvastatin subjected to CLR enrichment in vitro (n = 8). (“n” refers to number of artery segments). Here and in C, *statistically significant difference; P
    Filipin Dye, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore lr disruptors filipin
    Effects of Oxo on ASMase and ADP-ribosylcyclase activity in CAMs in the absence or presence of LR disruptors. A : summarized ASMase activity of CAMs treated with Oxo (80 μM, 15 min) alone or with 20-min pretreatment of MCD (100 μM), <t>filipin</t>
    Lr Disruptors Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore prepared filipin solution
    TSF attenuated glomerular mesangial matrix deposition, and lipid and cholesterol accumulation in the renal tissues of db/db mice. (A) PAS staining (bar = 25 μm). (B) Oil Red O staining (bar = 50 μm). (C) <t>Filipin</t> cholesterol staining (bar = 25 μm). (D) Analysis with a colorimetric assay demonstrated that TSF decreased total cholesterol levels in the renal tissues of 22-week-old db/db mice. ** P
    Prepared Filipin Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Millipore sterol binding compound filipin
    Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with <t>filipin.</t> The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type
    Sterol Binding Compound Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore lipid raft inhibitor filipin iii
    Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with <t>filipin.</t> The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type
    Lipid Raft Inhibitor Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore filipin pbs
    Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with <t>filipin.</t> The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type
    Filipin Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore high performance liquid chromatography grade filipin iii
    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of <t>filipin</t> for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of <t>three</t> measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.
    High Performance Liquid Chromatography Grade Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Millipore caveoli
    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of <t>filipin</t> for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of <t>three</t> measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.
    Caveoli, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore endocytyic pathway inhibitors
    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of <t>filipin</t> for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of <t>three</t> measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.
    Endocytyic Pathway Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore cytochalasin d
    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of <t>filipin</t> for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of <t>three</t> measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore lovastatin
    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of <t>filipin</t> for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of <t>three</t> measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.
    Lovastatin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore digitonin
    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of <t>filipin</t> for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of <t>three</t> measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.
    Digitonin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 4482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore amiloride
    Difference in cellular internalization depending on SNP size. Time-dependent cellular uptake of 20- and 50-nm SNPs with HepG2 cells was monitored by ( A ) flow cytometry and ( B ) fluorescent microscopy. ( C ) These cellular uptake in HepG2 cells incubated with endocytosis inhibitors (Chlorpromazine, Filipin III, and <t>Amiloride)</t> was examined, compare with non-incubated cells. This result indicate that the internalization mechanism of the SNPs at 20-nm level is not consistent with differed from those of 50-nm in size. At least in the early stages of internalization, the 20-nm SNP were capable of are trafficking into the cells, regardless independently of endocytosis.
    Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore anti flipl c terminal antibody
    Difference in cellular internalization depending on SNP size. Time-dependent cellular uptake of 20- and 50-nm SNPs with HepG2 cells was monitored by ( A ) flow cytometry and ( B ) fluorescent microscopy. ( C ) These cellular uptake in HepG2 cells incubated with endocytosis inhibitors (Chlorpromazine, Filipin III, and <t>Amiloride)</t> was examined, compare with non-incubated cells. This result indicate that the internalization mechanism of the SNPs at 20-nm level is not consistent with differed from those of 50-nm in size. At least in the early stages of internalization, the 20-nm SNP were capable of are trafficking into the cells, regardless independently of endocytosis.
    Anti Flipl C Terminal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore triton x 100
    Difference in cellular internalization depending on SNP size. Time-dependent cellular uptake of 20- and 50-nm SNPs with HepG2 cells was monitored by ( A ) flow cytometry and ( B ) fluorescent microscopy. ( C ) These cellular uptake in HepG2 cells incubated with endocytosis inhibitors (Chlorpromazine, Filipin III, and <t>Amiloride)</t> was examined, compare with non-incubated cells. This result indicate that the internalization mechanism of the SNPs at 20-nm level is not consistent with differed from those of 50-nm in size. At least in the early stages of internalization, the 20-nm SNP were capable of are trafficking into the cells, regardless independently of endocytosis.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore dimethyl sulfoxide dmso
    Effects of dynasore and NH 4 Cl on GCRV entry . GCRV-JX01 virions at an MOI of 20 were adsorbed to CIK cells that had been pretreated with ( a ) <t>DMSO(condition</t> equal to the volume of dynasore used for the 50 μM), ( b ) 50 μM dynasore in DMSO or ( c ) 20 mM ammonium chloride (NH 4 Cl) in water. After 1 h of viral adsorption at 0 °C, warm medium with inhibitors described above was quickly added. At 30 min post-infection (mpi), non-internalized viruses were removed and washed three times with PBS. Then cells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 for 10 min at room temperature. The samples were labeled with an anti-GCRV VP5 polyantibody ( green ) and DAPI ( Blue ). Scale bars represent 20 μM
    Dimethyl Sulfoxide Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 16048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Kinetics and mechanism of reaction of S S diphenylsulfilimine with 1 fluoro 2 4 dinitrobenzene 1 chloro 2 4 dinitrobenzene 2 chloro 3 nitropyridine 2 chloro 5 nitropyridine and 2
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    Image Search Results


    The cofilin and actin mutants facilitate the enrichment of sterols at the sites of tombusvirus replication in yeast. (A) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA. Fluorescent microscopic images of yeast cells stained with filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free wt yeast cells, while act1-121 ts yeast and act1-132 ts yeast show uneven distribution of ergosterols in the absence of viral components (right images). (B) Similar experiments as in Panel A, except performed at the semi-permissive temperature (27°C) in act1-132 ts and WT yeasts. (C) Similar experiments as in Panel A, except performed at the semi-permissive temperature (32°C) in act1-121 ts and WT yeasts. (D) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the permissive temperature. Fluorescent microscopic images of yeast cells stained with filipin dye. (E) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the semi-permissive temperature. See further details in panel A. Each experiment was repeated at least three times.

    Journal: PLoS Pathogens

    Article Title: Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    doi: 10.1371/journal.ppat.1005440

    Figure Lengend Snippet: The cofilin and actin mutants facilitate the enrichment of sterols at the sites of tombusvirus replication in yeast. (A) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA. Fluorescent microscopic images of yeast cells stained with filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free wt yeast cells, while act1-121 ts yeast and act1-132 ts yeast show uneven distribution of ergosterols in the absence of viral components (right images). (B) Similar experiments as in Panel A, except performed at the semi-permissive temperature (27°C) in act1-132 ts and WT yeasts. (C) Similar experiments as in Panel A, except performed at the semi-permissive temperature (32°C) in act1-121 ts and WT yeasts. (D) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the permissive temperature. Fluorescent microscopic images of yeast cells stained with filipin dye. (E) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the semi-permissive temperature. See further details in panel A. Each experiment was repeated at least three times.

    Article Snippet: Washed cells were incubated with 5 mg/ml filipin complex (Sigma Chemicals) in the dark for 15 min at 23°.

    Techniques: Staining

    Filipin staining of various C. albicans strains. All strains were grown to exponential phase in YEPD at 30°C. Cells were then transferred to serum containing medium and grown at 37°C. After shift to serum, cells were fixed at the indicated

    Journal: Fungal Genetics and Biology

    Article Title: Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi

    doi: 10.1016/j.fgb.2011.11.006

    Figure Lengend Snippet: Filipin staining of various C. albicans strains. All strains were grown to exponential phase in YEPD at 30°C. Cells were then transferred to serum containing medium and grown at 37°C. After shift to serum, cells were fixed at the indicated

    Article Snippet: Cells were incubated with 10 µg/ml filipin complex for S.cerevisiae and 5 µg/ml filipin complex (Sigma Chemicals) for C. albicans (Sigma Chemicals) in the dark for 15 min at 23°C.

    Techniques: Staining

    Electron microscopical visualization of filipin labeled membrane cholesterol. Arrowheads in control (A) and double-deficient fibroblasts (B) indicate the filipin labeling. In control cells labeling was detected in the limiting membranes of small vesicles

    Journal: Molecular Biology of the Cell

    Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts D⃞

    doi: 10.1091/mbc.E04-02-0103

    Figure Lengend Snippet: Electron microscopical visualization of filipin labeled membrane cholesterol. Arrowheads in control (A) and double-deficient fibroblasts (B) indicate the filipin labeling. In control cells labeling was detected in the limiting membranes of small vesicles

    Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS.

    Techniques: Labeling

    Reduction of cholesterol storage in LAMP double-deficient cells after LAMP-2a re-expression. (A and B) LAMP-2a was transiently transfected to LAMP1/LAMP-2 double-deficient cells. After 1 day, cholesterol was stained with filipin (A) and LAMP-2 was detected

    Journal: Molecular Biology of the Cell

    Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts D⃞

    doi: 10.1091/mbc.E04-02-0103

    Figure Lengend Snippet: Reduction of cholesterol storage in LAMP double-deficient cells after LAMP-2a re-expression. (A and B) LAMP-2a was transiently transfected to LAMP1/LAMP-2 double-deficient cells. After 1 day, cholesterol was stained with filipin (A) and LAMP-2 was detected

    Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS.

    Techniques: Expressing, Transfection, Staining

    Altered NPC1 localization and Nile Red staining in LAMP-1/LAMP-2 double-deficient cells. NPC1-GFP was transiently expressed in control cells (A) and in LAMP-1/LAMP-2 double-deficient cells (B). An overlay of NPC1-GFP (green) and filipin (red) is shown.

    Journal: Molecular Biology of the Cell

    Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts D⃞

    doi: 10.1091/mbc.E04-02-0103

    Figure Lengend Snippet: Altered NPC1 localization and Nile Red staining in LAMP-1/LAMP-2 double-deficient cells. NPC1-GFP was transiently expressed in control cells (A) and in LAMP-1/LAMP-2 double-deficient cells (B). An overlay of NPC1-GFP (green) and filipin (red) is shown.

    Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS.

    Techniques: Staining

    Cholesterol accumulation in LAMP-1/LAMP-2 double-deficient MEFs. Filipin staining revealed distribution of unesterified cholesterol in (A) control, (B) LAMP-1-/-, (C) LAMP-2-/-, and (D) LAMP-1/LAMP-2-/- cells. Note increased vesicular staining in LAMP-2

    Journal: Molecular Biology of the Cell

    Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts D⃞

    doi: 10.1091/mbc.E04-02-0103

    Figure Lengend Snippet: Cholesterol accumulation in LAMP-1/LAMP-2 double-deficient MEFs. Filipin staining revealed distribution of unesterified cholesterol in (A) control, (B) LAMP-1-/-, (C) LAMP-2-/-, and (D) LAMP-1/LAMP-2-/- cells. Note increased vesicular staining in LAMP-2

    Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS.

    Techniques: Staining

    STARD3 needs a functional START domain and ER–endosome contacts to induce cholesterol accumulation in endosomes Schematic representation of the different STARD3 mutants used in the study. The MENTAL domain in light blue contains 4 transmembrane helices (dark blue) and a FFAT motif (red); the START domain in pink contains two essential residues involved in cholesterol binding (M307 and N311). Positions of the epitopes recognized by the rabbit polyclonal pAbMLN64‐Nt‐1611 and pAbMLN64‐Ct‐605 antibodies are shown. Point mutation positions are labeled in black. Western blot analysis of STARD3 expression in the different cell lines. The expression of VAP proteins is unchanged; actin was used as a loading control. *: unspecific band. To mark cholesterol accumulation in endosomes, HeLa/Ctrl (a–c), HeLa/STARD3 (d–f), HeLa/STARD3 ΔSTART (g–i), HeLa/STARD3 MR/ND (j–l), and HeLa/STARD3 FA/YA (m–o) were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (C: green) or filipin (D: Cyan Hot). Nuclei are stained in blue. Higher magnification (2.5×) images of the area outlined in white (a, d, g, j, m) are shown on the right. The GFP‐D4 and STARD3 merged image (C) and the filipin and STARD3 merged image (D) are labeled Overlay. Data information: Scale bars: 10 μm.

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: STARD3 needs a functional START domain and ER–endosome contacts to induce cholesterol accumulation in endosomes Schematic representation of the different STARD3 mutants used in the study. The MENTAL domain in light blue contains 4 transmembrane helices (dark blue) and a FFAT motif (red); the START domain in pink contains two essential residues involved in cholesterol binding (M307 and N311). Positions of the epitopes recognized by the rabbit polyclonal pAbMLN64‐Nt‐1611 and pAbMLN64‐Ct‐605 antibodies are shown. Point mutation positions are labeled in black. Western blot analysis of STARD3 expression in the different cell lines. The expression of VAP proteins is unchanged; actin was used as a loading control. *: unspecific band. To mark cholesterol accumulation in endosomes, HeLa/Ctrl (a–c), HeLa/STARD3 (d–f), HeLa/STARD3 ΔSTART (g–i), HeLa/STARD3 MR/ND (j–l), and HeLa/STARD3 FA/YA (m–o) were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (C: green) or filipin (D: Cyan Hot). Nuclei are stained in blue. Higher magnification (2.5×) images of the area outlined in white (a, d, g, j, m) are shown on the right. The GFP‐D4 and STARD3 merged image (C) and the filipin and STARD3 merged image (D) are labeled Overlay. Data information: Scale bars: 10 μm.

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

    Techniques: Functional Assay, Binding Assay, Mutagenesis, Labeling, Western Blot, Expressing, Staining

    VAP protein knockdown abolishes STARD3‐mediated cholesterol accumulation in endosomes Western blot analysis of VAP‐A and VAP‐B expression in untreated (NT) HeLa/STARD3 cells or HeLa/STARD3 expressing a control shRNA (shCtrl) or two pairs of shRNAs targeting VAP‐A and VAP‐B (shVAP‐A/B‐α or shVAP‐A/B‐β). Actin was used as a loading control. HeLa cells (a–c), and HeLa/STARD3 cells expressing a control shRNA (shCtrl; d–f) or two pairs of shRNAs targeting VAP‐A and VAP‐B [shVAP‐A/B‐α (g–i) or shVAP‐A/B‐β (j–l)] were labeled with anti‐STARD3 antibodies (magenta) and with the fluorescent cholesterol probe filipin (Cyan Hot). Merged images of filipin and STARD3 signals are shown in (c, f, i and l). Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin signal in HeLa, HeLa/STARD3/shCtrl, HeLa/STARD3/shVAP‐A/B‐α, and HeLa/STARD3/shVAP‐A/B‐β. n = 3 independent experiments. Total number of cells analyzed: HeLa: 84; HeLa/STARD3/shCtrl: 130; HeLa/STARD3/shVAP‐A/B‐α: 109; HeLa/STARD3/shVAP‐A/B‐β: 92. Number of cells analyzed per sample per experiment ≥ 25. Mean ± SD; *** P

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: VAP protein knockdown abolishes STARD3‐mediated cholesterol accumulation in endosomes Western blot analysis of VAP‐A and VAP‐B expression in untreated (NT) HeLa/STARD3 cells or HeLa/STARD3 expressing a control shRNA (shCtrl) or two pairs of shRNAs targeting VAP‐A and VAP‐B (shVAP‐A/B‐α or shVAP‐A/B‐β). Actin was used as a loading control. HeLa cells (a–c), and HeLa/STARD3 cells expressing a control shRNA (shCtrl; d–f) or two pairs of shRNAs targeting VAP‐A and VAP‐B [shVAP‐A/B‐α (g–i) or shVAP‐A/B‐β (j–l)] were labeled with anti‐STARD3 antibodies (magenta) and with the fluorescent cholesterol probe filipin (Cyan Hot). Merged images of filipin and STARD3 signals are shown in (c, f, i and l). Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin signal in HeLa, HeLa/STARD3/shCtrl, HeLa/STARD3/shVAP‐A/B‐α, and HeLa/STARD3/shVAP‐A/B‐β. n = 3 independent experiments. Total number of cells analyzed: HeLa: 84; HeLa/STARD3/shCtrl: 130; HeLa/STARD3/shVAP‐A/B‐α: 109; HeLa/STARD3/shVAP‐A/B‐β: 92. Number of cells analyzed per sample per experiment ≥ 25. Mean ± SD; *** P

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

    Techniques: Western Blot, Expressing, shRNA, Labeling, Fluorescence

    Cholesterol staining with GFP‐D4 or filipin Plasma membrane cholesterol staining with the GFP‐D4 probe. Live HeLa/Ctrl cells were left untreated (a) or treated with MβCD (b) to remove plasma membrane cholesterol (10 mM in serum‐free medium; 30 min at 37°C), and incubated with GFP‐D4 prior to fixation and nucleus staining (blue). GFP‐D4 highly stained the plasma membrane of untreated cells (a), while almost no staining was present on MβCD‐treated cells (b). Analysis by flow cytometry of plasma cholesterol membrane staining with the GFP‐D4 probe. HeLa cells were either left untreated and unstained (HeLa/no probe), untreated (HeLa + GFP‐D4 probe) or treated with MβCD (HeLa/MβCD treatment + GFP‐D4 probe), and next stained; cells were then analyzed by flow cytometry. These representative histograms display the number of cells analyzed (normalized to mode) as a function of GFP‐D4 fluorescence (log intensity). Note that HeLa cells are strongly labeled with the GFP‐D4 probe; MβCD treatment prior to labeling strongly decreases GFP‐D4 signal intensity. Intracellular cholesterol staining with GFP‐D4. HeLa/Ctrl cells were left untreated (a) or treated with U18666A (1 μg/ml; 1 h at 37°C) to promote intracellular cholesterol accumulation (b). After fixation, cells were permeabilized by freezing in liquid nitrogen and incubated with GFP‐D4. Untreated cells (a) were labeled on small discrete structures by the GFP‐D4 probe; in U18666A‐treated cells (b), cholesterol‐filled endosomes were strongly labeled by the GFP‐D4 probe. Whole‐cell cholesterol staining with filipin on fixed HeLa/Ctrl (a) and HeLa/STARD3 (b) cells. Filipin stains cholesterol in the plasma membrane and in intracellular compartments. Note that HeLa/STARD3 cells display intracellular puncta of filipin staining. Intracellular cholesterol staining with filipin. Live HeLa/Ctrl (a) and HeLa/STARD3 (b) cells were treated with MβCD (10 mM in serum‐free medium; 30 min at 37°C) prior to fixation and filipin staining. MβCD treatment removed cholesterol from the plasma membrane and allowed a better visualization of intracellular cholesterol pools. Data information: Higher magnification (3×) images of the area outlined in white are shown on the right. Scale bars: 10 μm.

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: Cholesterol staining with GFP‐D4 or filipin Plasma membrane cholesterol staining with the GFP‐D4 probe. Live HeLa/Ctrl cells were left untreated (a) or treated with MβCD (b) to remove plasma membrane cholesterol (10 mM in serum‐free medium; 30 min at 37°C), and incubated with GFP‐D4 prior to fixation and nucleus staining (blue). GFP‐D4 highly stained the plasma membrane of untreated cells (a), while almost no staining was present on MβCD‐treated cells (b). Analysis by flow cytometry of plasma cholesterol membrane staining with the GFP‐D4 probe. HeLa cells were either left untreated and unstained (HeLa/no probe), untreated (HeLa + GFP‐D4 probe) or treated with MβCD (HeLa/MβCD treatment + GFP‐D4 probe), and next stained; cells were then analyzed by flow cytometry. These representative histograms display the number of cells analyzed (normalized to mode) as a function of GFP‐D4 fluorescence (log intensity). Note that HeLa cells are strongly labeled with the GFP‐D4 probe; MβCD treatment prior to labeling strongly decreases GFP‐D4 signal intensity. Intracellular cholesterol staining with GFP‐D4. HeLa/Ctrl cells were left untreated (a) or treated with U18666A (1 μg/ml; 1 h at 37°C) to promote intracellular cholesterol accumulation (b). After fixation, cells were permeabilized by freezing in liquid nitrogen and incubated with GFP‐D4. Untreated cells (a) were labeled on small discrete structures by the GFP‐D4 probe; in U18666A‐treated cells (b), cholesterol‐filled endosomes were strongly labeled by the GFP‐D4 probe. Whole‐cell cholesterol staining with filipin on fixed HeLa/Ctrl (a) and HeLa/STARD3 (b) cells. Filipin stains cholesterol in the plasma membrane and in intracellular compartments. Note that HeLa/STARD3 cells display intracellular puncta of filipin staining. Intracellular cholesterol staining with filipin. Live HeLa/Ctrl (a) and HeLa/STARD3 (b) cells were treated with MβCD (10 mM in serum‐free medium; 30 min at 37°C) prior to fixation and filipin staining. MβCD treatment removed cholesterol from the plasma membrane and allowed a better visualization of intracellular cholesterol pools. Data information: Higher magnification (3×) images of the area outlined in white are shown on the right. Scale bars: 10 μm.

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

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

    The ER is the main source of cholesterol accumulated by STARD3 in endosomes HeLa/Ctrl and HeLa/STARD3 cells were incubated in normal medium (A), LPDS‐containing medium (B) or normal medium with 50 μM mevinolin and 100 μM mevalonate (C), for 48 h. Cholesterol accumulation in endosomes was detected by filipin staining (Cyan Hot) in endosomes identified by the presence of Lamp1 (red) and STARD3 (magenta). Nuclei were stained in blue. Merged image of filipin and STARD3 signals is shown in (d and h). The subpanels on the right are higher magnification (3.5×) images of the area outlined in white (a, e). The filipin and STARD3 merged image is labeled Overlay. Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin in HeLa/Ctrl and HeLa/STARD3 cells incubated or not in LPDS‐containing medium (D) or treated or not with mevinolin and mevalonate (E). Mean ± SD; n = 5 (D) and n = 4 (E) independent experiments; ** P

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: The ER is the main source of cholesterol accumulated by STARD3 in endosomes HeLa/Ctrl and HeLa/STARD3 cells were incubated in normal medium (A), LPDS‐containing medium (B) or normal medium with 50 μM mevinolin and 100 μM mevalonate (C), for 48 h. Cholesterol accumulation in endosomes was detected by filipin staining (Cyan Hot) in endosomes identified by the presence of Lamp1 (red) and STARD3 (magenta). Nuclei were stained in blue. Merged image of filipin and STARD3 signals is shown in (d and h). The subpanels on the right are higher magnification (3.5×) images of the area outlined in white (a, e). The filipin and STARD3 merged image is labeled Overlay. Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin in HeLa/Ctrl and HeLa/STARD3 cells incubated or not in LPDS‐containing medium (D) or treated or not with mevinolin and mevalonate (E). Mean ± SD; n = 5 (D) and n = 4 (E) independent experiments; ** P

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

    Techniques: Incubation, Staining, Labeling, Fluorescence

    Characterization of cholesterol‐enriched endosomes in HeLa/STARD3 cells HeLa/STARD3 cells were co‐labeled with anti‐STARD3 (magenta), with the fluorescent cholesterol probe filipin (Cyan Hot) and with anti‐EEA1 (a–d, green), anti‐Lamp1 (e–h, green), anti‐CD63 (i–l, green), anti‐Rab7 (m–p, green), or anti‐BMP (q–t, green) antibodies. Nuclei were stained in blue. Merged image of magenta and green signals is shown in (c, g, k, o, and s). The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (c, g, k, o, s). Overlays show STARD3 and the endocytic markers merged images. Scale bars: 10 μm. Pearson correlation coefficients between STARD3 and the endocytic markers EEA1, Lamp1, CD63, Rab7, and BMP are shown; each dot represents one single cell; cells originates from three independent experiments (15 cells). The horizontal lines show the mean ± SD. Pearson correlation coefficients between filipin and the early endosome marker EEA1 (left) and with the late endosome marker Rab7 (right). Each dot represents one single cell (left 15 cells, right: 20 cells); cells originate from three independent experiments. The horizontal lines show the mean ± SD. HeLa/STARD3 cells were co‐stained with two fluorescent cholesterol probes (GFP‐D4 in green and filipin in Cyan Hot) and with anti‐STARD3 (magenta). Two similar cells are shown (bottom and top). Nuclei were stained in blue. (d, h) Pixels where the green and the Cyan Hot channels co‐localize are shown in white. The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (a, e). Scale bars: 10 μm. Mander's correlation coefficients between filipin and GFP‐D4 are shown; each dot represents one single cell acquired from three independent experiments (22 cells). M1 corresponds to the fraction of filipin signal overlapping with GFP‐D4 signal. M2 corresponds to the fraction of GFP‐D4 signal overlapping with filipin signal. GFP‐D4‐labeled structures are strongly labeled with filipin, while filipin‐positive structures can be GFP‐D4 positive or GFP‐D4 negative. This illustrates the different properties of the two cholesterol probes: While filipin binds to all free cholesterol, GFP‐D4 only binds to cholesterol‐rich membranes. The horizontal lines show the mean ± SD. Paired two‐tailed t ‐test; *** P

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: Characterization of cholesterol‐enriched endosomes in HeLa/STARD3 cells HeLa/STARD3 cells were co‐labeled with anti‐STARD3 (magenta), with the fluorescent cholesterol probe filipin (Cyan Hot) and with anti‐EEA1 (a–d, green), anti‐Lamp1 (e–h, green), anti‐CD63 (i–l, green), anti‐Rab7 (m–p, green), or anti‐BMP (q–t, green) antibodies. Nuclei were stained in blue. Merged image of magenta and green signals is shown in (c, g, k, o, and s). The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (c, g, k, o, s). Overlays show STARD3 and the endocytic markers merged images. Scale bars: 10 μm. Pearson correlation coefficients between STARD3 and the endocytic markers EEA1, Lamp1, CD63, Rab7, and BMP are shown; each dot represents one single cell; cells originates from three independent experiments (15 cells). The horizontal lines show the mean ± SD. Pearson correlation coefficients between filipin and the early endosome marker EEA1 (left) and with the late endosome marker Rab7 (right). Each dot represents one single cell (left 15 cells, right: 20 cells); cells originate from three independent experiments. The horizontal lines show the mean ± SD. HeLa/STARD3 cells were co‐stained with two fluorescent cholesterol probes (GFP‐D4 in green and filipin in Cyan Hot) and with anti‐STARD3 (magenta). Two similar cells are shown (bottom and top). Nuclei were stained in blue. (d, h) Pixels where the green and the Cyan Hot channels co‐localize are shown in white. The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (a, e). Scale bars: 10 μm. Mander's correlation coefficients between filipin and GFP‐D4 are shown; each dot represents one single cell acquired from three independent experiments (22 cells). M1 corresponds to the fraction of filipin signal overlapping with GFP‐D4 signal. M2 corresponds to the fraction of GFP‐D4 signal overlapping with filipin signal. GFP‐D4‐labeled structures are strongly labeled with filipin, while filipin‐positive structures can be GFP‐D4 positive or GFP‐D4 negative. This illustrates the different properties of the two cholesterol probes: While filipin binds to all free cholesterol, GFP‐D4 only binds to cholesterol‐rich membranes. The horizontal lines show the mean ± SD. Paired two‐tailed t ‐test; *** P

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

    Techniques: Labeling, Staining, Marker, Two Tailed Test

    Image analysis procedure for filipin staining intensity quantification Fields containing about a dozen of cells labeled with filipin, anti‐STARD3, and propidium iodide (PI) were randomly acquired by confocal microscopy in three‐channel images. Image analysis was performed with Fiji ( http://fiji.sc/ ). Image segmentation was performed on the PI staining image. An intensity threshold was applied to determine the cell contours. Nuclei positions, manually selected on the PI staining image, were used to divide the image into discrete areas with a watershed algorithm (Find maxima). Combination of the thresholded and the segmented particles images allowed to build a segmentation mask where individual cell contours could be determined. To quantify the filipin staining intensity, an intensity threshold was first applied on raw images. This threshold allowed to focus the analysis on filipin accumulation puncta. After applying the segmentation mask onto the filipin staining thresholded image, filipin staining intensity was measured in individual cells.

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: Image analysis procedure for filipin staining intensity quantification Fields containing about a dozen of cells labeled with filipin, anti‐STARD3, and propidium iodide (PI) were randomly acquired by confocal microscopy in three‐channel images. Image analysis was performed with Fiji ( http://fiji.sc/ ). Image segmentation was performed on the PI staining image. An intensity threshold was applied to determine the cell contours. Nuclei positions, manually selected on the PI staining image, were used to divide the image into discrete areas with a watershed algorithm (Find maxima). Combination of the thresholded and the segmented particles images allowed to build a segmentation mask where individual cell contours could be determined. To quantify the filipin staining intensity, an intensity threshold was first applied on raw images. This threshold allowed to focus the analysis on filipin accumulation puncta. After applying the segmentation mask onto the filipin staining thresholded image, filipin staining intensity was measured in individual cells.

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

    Techniques: Staining, Labeling, Confocal Microscopy

    STARD3 favors cholesterol accumulation in endosomes To follow cholesterol accumulation in late endosomes, HeLa (a–d), HeLa/Ctrl (e–h), and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (green). Nuclei were stained in blue. Merged image of GFP‐D4 and STARD3 signals is shown in (d, h, and l). The subpanels on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). Overlay indicates GFP‐D4 and STARD3 merged image. (n) Linescan analyses with relative fluorescence intensities of the green, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes (LE). Colocalization between GFP‐D4‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (12 cells). Filipin as a second method to follow cholesterol accumulation in late endosomes. HeLa (a–d), HeLa/Ctrl (e–h) and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are stained in blue. Merged image of filipin and STARD3 signals is shown in (d, h and l). Shown on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). The filipin and STARD3 merged image is labeled Overlay. (n) Linescan analyses with relative fluorescence intensities of the cyan, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes. Colocalization between filipin‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (10 cells). Pearson correlation coefficients between STARD3 and GFP‐D4 (left) or filipin (right) staining are shown. Each dot represents a single cell (GFP‐D4: 16 cells; filipin: 24 cells; from three independent experiments). Relative fluorescence intensity of intracellular filipin signals in HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART, HeLa/STARD3 MR/ND, and HeLa/STARD3 FA/YA cells. n : number of independent experiments. HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART: n = 6; HeLa/STARD3 MR/ND and HeLa/STARD3 FA/YA: n = 3. Total number of cells analyzed: HeLa: 309; HeLa/Ctrl: 234; HeLa/STARD3: 295; HeLa/STARD3 ΔSTART: 238; HeLa/STARD3 MR/ND: 116 and HeLa/STARD3 FA/YA: 137. Number of cells analyzed per sample per experiment ≥ 32. Data information: Scale bars: 10 μm. Mean ± SD; * P

    Journal: The EMBO Journal

    Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

    doi: 10.15252/embj.201695917

    Figure Lengend Snippet: STARD3 favors cholesterol accumulation in endosomes To follow cholesterol accumulation in late endosomes, HeLa (a–d), HeLa/Ctrl (e–h), and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (green). Nuclei were stained in blue. Merged image of GFP‐D4 and STARD3 signals is shown in (d, h, and l). The subpanels on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). Overlay indicates GFP‐D4 and STARD3 merged image. (n) Linescan analyses with relative fluorescence intensities of the green, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes (LE). Colocalization between GFP‐D4‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (12 cells). Filipin as a second method to follow cholesterol accumulation in late endosomes. HeLa (a–d), HeLa/Ctrl (e–h) and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are stained in blue. Merged image of filipin and STARD3 signals is shown in (d, h and l). Shown on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). The filipin and STARD3 merged image is labeled Overlay. (n) Linescan analyses with relative fluorescence intensities of the cyan, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes. Colocalization between filipin‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (10 cells). Pearson correlation coefficients between STARD3 and GFP‐D4 (left) or filipin (right) staining are shown. Each dot represents a single cell (GFP‐D4: 16 cells; filipin: 24 cells; from three independent experiments). Relative fluorescence intensity of intracellular filipin signals in HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART, HeLa/STARD3 MR/ND, and HeLa/STARD3 FA/YA cells. n : number of independent experiments. HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART: n = 6; HeLa/STARD3 MR/ND and HeLa/STARD3 FA/YA: n = 3. Total number of cells analyzed: HeLa: 309; HeLa/Ctrl: 234; HeLa/STARD3: 295; HeLa/STARD3 ΔSTART: 238; HeLa/STARD3 MR/ND: 116 and HeLa/STARD3 FA/YA: 137. Number of cells analyzed per sample per experiment ≥ 32. Data information: Scale bars: 10 μm. Mean ± SD; * P

    Article Snippet: Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

    Techniques: Labeling, Staining, Fluorescence

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0029537

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

    Article Snippet: After extensive washing, cells were incubated with a 10 µM Filipin III (Streptomyces filipinensis , Sigma-Aldrich) solution in PBS for 30 min at room temperature.

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0029537

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

    Article Snippet: After extensive washing, cells were incubated with a 10 µM Filipin III (Streptomyces filipinensis , Sigma-Aldrich) solution in PBS for 30 min at room temperature.

    Techniques: Fluorescence, Staining, Cell Culture, Incubation

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

    Journal: Journal of Neurophysiology

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

    doi: 10.1152/jn.00779.2011

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

    Article Snippet: To image membrane lipids at the terminals of cone photoreceptors, we applied filipin III (Sigma-Aldrich) to label membrane cholesterol or FITC-CTXB (Sigma-Aldrich) to label ganglioside GM1 glycoproteins associated with cholesterol-rich lipid rafts.

    Techniques: Staining

    Effect of inhibitors on internalization of A. actinomycetemcomitans OMVs in HeLa cells. (A) HeLa cells pretreated for 30 min with monensin (+Mo) (10 μM), or filipin III (+Fi) (10 μg/ml) were incubated with PKH26-labeled OMVs for 24 h.

    Journal: Infection and Immunity

    Article Title: Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

    doi: 10.1128/IAI.01980-14

    Figure Lengend Snippet: Effect of inhibitors on internalization of A. actinomycetemcomitans OMVs in HeLa cells. (A) HeLa cells pretreated for 30 min with monensin (+Mo) (10 μM), or filipin III (+Fi) (10 μg/ml) were incubated with PKH26-labeled OMVs for 24 h.

    Article Snippet: Where applicable, vesicles were incubated with cells in the presence of the inhibiting agent filipin III (Sigma-Aldrich) at a final concentration of 10 μg/ml as in earlier studies ( , , ).

    Techniques: Incubation, Labeling

    GP73 regulates the transcriptional activity of SREBPs and lipogenesis. ( a ) Immunoblotting analysis of SREBPs activation in HepG2 cells transfected with Flag-GP73 at the indicated doses. α-Tubulin was used as equal loading control. ( b , c ) SREBP-1 promoter activity in HepG2 ( b ) or HL7702 ( c ) cells transfected with Flag-vector or Flag-GP73 under conditions of sterol depletion or repletion. The luciferase activity was measured 36 hrs post transfection. The value was normalized with the corresponding transfection efficiency. ( d , f , h ) QRT-PCR analysis of HMGR ( d ), FASN2 ( f ), and ACC1 ( h ) mRNA abundance in HepG2 cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( e , g , i ) QRT-PCR analysis of HMGR ( e ), FASN2 ( g ), and ACC1 ( i ) mRNA abundance in 293T cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( j ) Fluorescence microscopy of Filipin staining in HepG2 cells transfected with Flag-GP73. Cells were collected at indicated hrs post transfection. ( k , l ) Amplex Red cholesterol assay of cellular cholesterol concentrations in HepG2 ( k ) or HL7702 ( l ) cells transfected with Flag-vector or Flag-GP73. Cells were collected at indicated hrs post transfection. Values were normalized to total cell proteins from control cells transfected with Flag-vector. Cell-based studies were performed at least three independent times with comparable results. Data represent mean ± SEM. Student’s t test was used for statistical analysis: **p

    Journal: Scientific Reports

    Article Title: GP73 regulates Hepatic Steatosis by enhancing SCAP-SREBPs interaction

    doi: 10.1038/s41598-017-06500-9

    Figure Lengend Snippet: GP73 regulates the transcriptional activity of SREBPs and lipogenesis. ( a ) Immunoblotting analysis of SREBPs activation in HepG2 cells transfected with Flag-GP73 at the indicated doses. α-Tubulin was used as equal loading control. ( b , c ) SREBP-1 promoter activity in HepG2 ( b ) or HL7702 ( c ) cells transfected with Flag-vector or Flag-GP73 under conditions of sterol depletion or repletion. The luciferase activity was measured 36 hrs post transfection. The value was normalized with the corresponding transfection efficiency. ( d , f , h ) QRT-PCR analysis of HMGR ( d ), FASN2 ( f ), and ACC1 ( h ) mRNA abundance in HepG2 cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( e , g , i ) QRT-PCR analysis of HMGR ( e ), FASN2 ( g ), and ACC1 ( i ) mRNA abundance in 293T cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( j ) Fluorescence microscopy of Filipin staining in HepG2 cells transfected with Flag-GP73. Cells were collected at indicated hrs post transfection. ( k , l ) Amplex Red cholesterol assay of cellular cholesterol concentrations in HepG2 ( k ) or HL7702 ( l ) cells transfected with Flag-vector or Flag-GP73. Cells were collected at indicated hrs post transfection. Values were normalized to total cell proteins from control cells transfected with Flag-vector. Cell-based studies were performed at least three independent times with comparable results. Data represent mean ± SEM. Student’s t test was used for statistical analysis: **p

    Article Snippet: For Filipin staining, cells grown on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by 2 hrs incubation in a freshly prepared Filipin III (Sigma) solution (50 μg/ml).

    Techniques: Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Fluorescence, Microscopy, Staining, Amplex Red Cholesterol Assay

    Fluorescence imaging confirms differences in MCA smooth muscle CLR levels among experimental groups. A. Original snapshots showing vascular smooth muscle CLR levels via fluorescence staining of arteries with CLR-sensitive dye filipin. Arteries were collected from rats on high CLR diet + placebo, and high CLR diet + atorvastatin. Several arteries from rats on high CLR diet + atorvastatin were subjected to in vitro CLR enrichment. Artery staining from all experimental groups was performed in parallel to minimize the experimental error and justify the direct comparison of the fluorescence signal intensity. Snapshots on the right show smooth muscle layer in visible light. These images were superposed with fluorescence ones to define the individual myocyte plasma membranes for filipin intensity quantification. In the right top panel, arrows point at the example of individual myocyte’s silhouette (light grey) within vasculature layer. B. Averaged data showing filipin fluorescence signal from high CLR diet + placebo (n = 7), high CLR diet + atorvastatin (n = 10), and high CLR diet + atorvastatin subjected to CLR enrichment in vitro (n = 8). (“n” refers to number of artery segments). Here and in C, *statistically significant difference; P

    Journal: Biochemical pharmacology

    Article Title: Statin therapy exacerbates alcohol-induced constriction of cerebral arteries via modulation of ethanol-induced BK channel inhibition in vascular smooth muscle

    doi: 10.1016/j.bcp.2017.08.022

    Figure Lengend Snippet: Fluorescence imaging confirms differences in MCA smooth muscle CLR levels among experimental groups. A. Original snapshots showing vascular smooth muscle CLR levels via fluorescence staining of arteries with CLR-sensitive dye filipin. Arteries were collected from rats on high CLR diet + placebo, and high CLR diet + atorvastatin. Several arteries from rats on high CLR diet + atorvastatin were subjected to in vitro CLR enrichment. Artery staining from all experimental groups was performed in parallel to minimize the experimental error and justify the direct comparison of the fluorescence signal intensity. Snapshots on the right show smooth muscle layer in visible light. These images were superposed with fluorescence ones to define the individual myocyte plasma membranes for filipin intensity quantification. In the right top panel, arrows point at the example of individual myocyte’s silhouette (light grey) within vasculature layer. B. Averaged data showing filipin fluorescence signal from high CLR diet + placebo (n = 7), high CLR diet + atorvastatin (n = 10), and high CLR diet + atorvastatin subjected to CLR enrichment in vitro (n = 8). (“n” refers to number of artery segments). Here and in C, *statistically significant difference; P

    Article Snippet: Middle cerebral arteries were fixed in 3% paraformaldehyde at room temperature for 30 min and permeabilized with 0.1% Triton-100 in phosphate buffered saline at room temperature for 30 min. For filipin staining, arteries were incubated with the CLR-sensitive dye filipin (Sigma, St. Louis, MO) at room temperature for 2 h. For immunostaining, arteries were incubated at room temperature for 2 h in a mixture of the following primary antibodies: mouse monoclonal antibody against BK alpha subunit (clone L6/60, UC Davis/NIH NeuroMab Facility, Davis, CA) and rabbit polyclonal antibody against BK beta 1 subunit (PA 1–924, Invitrogen, Carlsbad, CA).

    Techniques: Fluorescence, Imaging, Staining, In Vitro

    Filipin staining of M. tuberculosis grown with (A) or without (B) cholesterol and M. tuberculosis defatted cells grown in the presence of cholesterol (C). Cell morphology was visualized by differential interference contrast microscopy (left). Fluorescence

    Journal: Journal of Bacteriology

    Article Title: Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol ▿ Is Able To Accumulate and Utilize Cholesterol ▿ †

    doi: 10.1128/JB.00488-09

    Figure Lengend Snippet: Filipin staining of M. tuberculosis grown with (A) or without (B) cholesterol and M. tuberculosis defatted cells grown in the presence of cholesterol (C). Cell morphology was visualized by differential interference contrast microscopy (left). Fluorescence

    Article Snippet: In order to visualize cholesterol accumulation in the mycobacterial cells, we used the fluorescent dye filipin (Sigma-Aldrich).

    Techniques: Staining, Microscopy, Fluorescence

    Effects of Oxo on ASMase and ADP-ribosylcyclase activity in CAMs in the absence or presence of LR disruptors. A : summarized ASMase activity of CAMs treated with Oxo (80 μM, 15 min) alone or with 20-min pretreatment of MCD (100 μM), filipin

    Journal:

    Article Title: Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes

    doi: 10.1152/ajpheart.00617.2008

    Figure Lengend Snippet: Effects of Oxo on ASMase and ADP-ribosylcyclase activity in CAMs in the absence or presence of LR disruptors. A : summarized ASMase activity of CAMs treated with Oxo (80 μM, 15 min) alone or with 20-min pretreatment of MCD (100 μM), filipin

    Article Snippet: In additional groups of cells, LR disruptors filipin (1 μg; Sigma) or methyl-β-cyclodextrin (MCD; 100 μM; Sigma) or an ASMase inhibitor desipramine (Des, 10 μM; Sigma) were added and incubated for 20 min before Oxo stimulation.

    Techniques: Activity Assay

    Western blot analysis of CD38 and ASMase in LR fractions isolated from CAMs stimulated by Oxo. A : CAMs were treated with Oxo (80 μM, 15 min) alone or with 20-min pretreatment of MCD (100 μM), filipin (1 μg), or Des (10 μM).

    Journal:

    Article Title: Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes

    doi: 10.1152/ajpheart.00617.2008

    Figure Lengend Snippet: Western blot analysis of CD38 and ASMase in LR fractions isolated from CAMs stimulated by Oxo. A : CAMs were treated with Oxo (80 μM, 15 min) alone or with 20-min pretreatment of MCD (100 μM), filipin (1 μg), or Des (10 μM).

    Article Snippet: In additional groups of cells, LR disruptors filipin (1 μg; Sigma) or methyl-β-cyclodextrin (MCD; 100 μM; Sigma) or an ASMase inhibitor desipramine (Des, 10 μM; Sigma) were added and incubated for 20 min before Oxo stimulation.

    Techniques: Western Blot, Isolation

    TSF attenuated glomerular mesangial matrix deposition, and lipid and cholesterol accumulation in the renal tissues of db/db mice. (A) PAS staining (bar = 25 μm). (B) Oil Red O staining (bar = 50 μm). (C) Filipin cholesterol staining (bar = 25 μm). (D) Analysis with a colorimetric assay demonstrated that TSF decreased total cholesterol levels in the renal tissues of 22-week-old db/db mice. ** P

    Journal: Frontiers in Physiology

    Article Title: Tangshen Formula Attenuates Diabetic Nephropathy by Promoting ABCA1-Mediated Renal Cholesterol Efflux in db/db Mice

    doi: 10.3389/fphys.2018.00343

    Figure Lengend Snippet: TSF attenuated glomerular mesangial matrix deposition, and lipid and cholesterol accumulation in the renal tissues of db/db mice. (A) PAS staining (bar = 25 μm). (B) Oil Red O staining (bar = 50 μm). (C) Filipin cholesterol staining (bar = 25 μm). (D) Analysis with a colorimetric assay demonstrated that TSF decreased total cholesterol levels in the renal tissues of 22-week-old db/db mice. ** P

    Article Snippet: For filipin cholesterol staining, sections were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, and then stained with freshly prepared filipin solution (125 μg/mL, Sigma-Aldrich) for 30 min. Next, the slides were washed with PBS, and a drop of glycerol was added.

    Techniques: Mouse Assay, Staining, Colorimetric Assay

    Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with filipin. The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type

    Journal: Development (Cambridge, England)

    Article Title: Survival strategies of a sterol auxotroph

    doi: 10.1242/dev.044560

    Figure Lengend Snippet: Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with filipin. The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type

    Article Snippet: Tissues were fixed and stained with the fluorescent sterol-binding compound filipin (Sigma) as described previously ( ) and mounted using VECTASHIELD mounting medium (Vector Laboratories).

    Techniques: Staining

    Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of filipin for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of three measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.

    Journal: International Journal of Nanomedicine

    Article Title: Delivery system for DNAzymes using arginine-modified hydroxyapatite nanoparticles for therapeutic application in a nasopharyngeal carcinoma model

    doi: 10.2147/IJN.S48321

    Figure Lengend Snippet: Cellular uptake of the Arg-nHAP/DZ1 complex in the presence of specific endocytic inhibitors. Cells were pretreated with inhibitors and transfected with the Arg-nHAP/FITC-DZ1 complex. Concentrations of the inhibitors are as follows: 20 mM sodium azide and 50 mM 2-deoxy-D-glucose for one hour; 0.15 μM PAO for 10 minutes; and 1.25 μg/mL of filipin for one hour. The effect of low temperature on cellular uptake was investigated at 4°C. All values are the mean of three measurements and are shown with error bars. Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; PAO, phenylarsine oxide; DZ1, DNAzyme 1.

    Article Snippet: Materials The chemicals, inhibitors, transfection reagents, and cell culture media used in these experiments were sourced as follows: fluorescein isothiocyanate (FITC)-labeled DZ1 (FITC-DZ1) and control DNAzyme (CON) were synthesized by Oligos Etc Inc (Portland, OR, USA); Lipofectamine™ 2000, ProLong® gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole), and trypsin-EDTA were from Invitrogen Life Technologies (Grand Island, NY, USA); high-performance liquid chromatography grade filipin III ( > 85%), phenylarsine oxide (≥97%), MTS (3-(4,5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and sodium azide were from Sigma-Aldrich (St Louis, MO, USA); 2-deoxy-D-glucose was from Tokyo Chemical Industry (Tokyo, Japan); Fugene HD was from Roche (Basel, Switzerland); and fetal bovine serum was from Gibco (Grand Island, NY, USA).

    Techniques: Transfection, Modification

    Difference in cellular internalization depending on SNP size. Time-dependent cellular uptake of 20- and 50-nm SNPs with HepG2 cells was monitored by ( A ) flow cytometry and ( B ) fluorescent microscopy. ( C ) These cellular uptake in HepG2 cells incubated with endocytosis inhibitors (Chlorpromazine, Filipin III, and Amiloride) was examined, compare with non-incubated cells. This result indicate that the internalization mechanism of the SNPs at 20-nm level is not consistent with differed from those of 50-nm in size. At least in the early stages of internalization, the 20-nm SNP were capable of are trafficking into the cells, regardless independently of endocytosis.

    Journal: International Journal of Nanomedicine

    Article Title: A reliable approach for assessing size-dependent effects of silica nanoparticles on cellular internalization behavior and cytotoxic mechanisms

    doi: 10.2147/IJN.S224183

    Figure Lengend Snippet: Difference in cellular internalization depending on SNP size. Time-dependent cellular uptake of 20- and 50-nm SNPs with HepG2 cells was monitored by ( A ) flow cytometry and ( B ) fluorescent microscopy. ( C ) These cellular uptake in HepG2 cells incubated with endocytosis inhibitors (Chlorpromazine, Filipin III, and Amiloride) was examined, compare with non-incubated cells. This result indicate that the internalization mechanism of the SNPs at 20-nm level is not consistent with differed from those of 50-nm in size. At least in the early stages of internalization, the 20-nm SNP were capable of are trafficking into the cells, regardless independently of endocytosis.

    Article Snippet: Reagents and antibodies Fluorescein isothiocyanate (FITC), (3-Aminopropyl) triethoxysilane (APTES), Chlorpromazine, Filipin III, and Amiloride were purchased from Sigma-Aldrich.

    Techniques: Flow Cytometry, Cytometry, Microscopy, Incubation

    Cellular internalization and cytotoxicity of SNP depending on serum concentration. ( A ) The difference in the cellular uptake of 20- and 50-nm SNPs into HEPG2 cells according to serum concentration was monitored by flow cytometry. Unlike the 50-nm SNP, 20-nm SNP in the 5% serum-containing condition were fully transferred into the cells. ( B ) Time-dependent cellular uptake of 20-nm SNPs with HepG2 cells was monitored by flow cytometry (5% serum condition). Filled histogram indicate untreated cells as control. Also, its cellular uptake in HepG2 cells incubated with/without endocytosis inhibitors (Chlorpromazine, Filipin III, and Amiloride) was examined comparatively. These SNPs appear to be time-dependent internalization behavior through the endocytosis pathway as if they were 50-nm SNPs. ( C, D ) 20-nm SNPs were treated into the cells as the predetermined serum concentration and exposure time of SNP. ( C ) Representative bar graph of the percentages of apoptotic and necrotic cells death as determined by flow cytometric analysis. ( D ) Interaction between RIPK1–RIPK3 was detected by immunoprecipitation (IP) and Western blot analysis. These results show that the 20-nm SNPs larger in size by serum are no longer induced by early necrosis.

    Journal: International Journal of Nanomedicine

    Article Title: A reliable approach for assessing size-dependent effects of silica nanoparticles on cellular internalization behavior and cytotoxic mechanisms

    doi: 10.2147/IJN.S224183

    Figure Lengend Snippet: Cellular internalization and cytotoxicity of SNP depending on serum concentration. ( A ) The difference in the cellular uptake of 20- and 50-nm SNPs into HEPG2 cells according to serum concentration was monitored by flow cytometry. Unlike the 50-nm SNP, 20-nm SNP in the 5% serum-containing condition were fully transferred into the cells. ( B ) Time-dependent cellular uptake of 20-nm SNPs with HepG2 cells was monitored by flow cytometry (5% serum condition). Filled histogram indicate untreated cells as control. Also, its cellular uptake in HepG2 cells incubated with/without endocytosis inhibitors (Chlorpromazine, Filipin III, and Amiloride) was examined comparatively. These SNPs appear to be time-dependent internalization behavior through the endocytosis pathway as if they were 50-nm SNPs. ( C, D ) 20-nm SNPs were treated into the cells as the predetermined serum concentration and exposure time of SNP. ( C ) Representative bar graph of the percentages of apoptotic and necrotic cells death as determined by flow cytometric analysis. ( D ) Interaction between RIPK1–RIPK3 was detected by immunoprecipitation (IP) and Western blot analysis. These results show that the 20-nm SNPs larger in size by serum are no longer induced by early necrosis.

    Article Snippet: Reagents and antibodies Fluorescein isothiocyanate (FITC), (3-Aminopropyl) triethoxysilane (APTES), Chlorpromazine, Filipin III, and Amiloride were purchased from Sigma-Aldrich.

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, Incubation, Immunoprecipitation, Western Blot

    Effects of dynasore and NH 4 Cl on GCRV entry . GCRV-JX01 virions at an MOI of 20 were adsorbed to CIK cells that had been pretreated with ( a ) DMSO(condition equal to the volume of dynasore used for the 50 μM), ( b ) 50 μM dynasore in DMSO or ( c ) 20 mM ammonium chloride (NH 4 Cl) in water. After 1 h of viral adsorption at 0 °C, warm medium with inhibitors described above was quickly added. At 30 min post-infection (mpi), non-internalized viruses were removed and washed three times with PBS. Then cells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 for 10 min at room temperature. The samples were labeled with an anti-GCRV VP5 polyantibody ( green ) and DAPI ( Blue ). Scale bars represent 20 μM

    Journal: Virology Journal

    Article Title: Disruption of clathrin-dependent trafficking results in the failure of grass carp reovirus cellular entry

    doi: 10.1186/s12985-016-0485-7

    Figure Lengend Snippet: Effects of dynasore and NH 4 Cl on GCRV entry . GCRV-JX01 virions at an MOI of 20 were adsorbed to CIK cells that had been pretreated with ( a ) DMSO(condition equal to the volume of dynasore used for the 50 μM), ( b ) 50 μM dynasore in DMSO or ( c ) 20 mM ammonium chloride (NH 4 Cl) in water. After 1 h of viral adsorption at 0 °C, warm medium with inhibitors described above was quickly added. At 30 min post-infection (mpi), non-internalized viruses were removed and washed three times with PBS. Then cells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 for 10 min at room temperature. The samples were labeled with an anti-GCRV VP5 polyantibody ( green ) and DAPI ( Blue ). Scale bars represent 20 μM

    Article Snippet: Chemicals Ammonium chloride (NH4 Cl), chloroquine (CQ), nystatin, Filipin III, and dimethyl sulfoxide (DMSO) were purchased from Sigma.

    Techniques: Adsorption, Infection, Labeling