filipin Millipore Search Results


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  • 99
    Millipore filipin
    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 <t>filipin</t> (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.
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore prepared filipin iii
    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
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    Millipore filipin reagent
    Cerebellar cortex, 15-month-old knockout mice. ( A ) Glycol methacrylate-embedded tissue sectioned at 3 μm, stained with Richardson’s stain. Purkinje neuron cell bodies (white arrows) are mildly vacuolated (and almost free of glycogen, as seen in B ), whereas the cell bodies of adjacent Golgi epithelial cells (radially-oriented astrocytes) are markedly vacuolated. Magnification: 200x; bar: 20 μm. a′. Cryostat section, 20-μm-thick stained with the fluorescent dye, <t>filipin,</t> shows intense accumulation of cholesterol in the apical cytoplasm of Purkinje neurons (white arrows), even though these cells differ from most other large neurons in not accumulating comparable amounts of glycogen ( B ). Magnification: 100x. ( B ) Paraffin section, 7 μm, stained with the PAS method and counterstained with hematoxylin. There is marked glycogen accumulation in the cell bodies of Golgi epithelial cell astrocytes (horizontal black arrows) and in clumps in the granular layer and in the cytoplasm of most glial cells in the cerebellar cortical white matter (oblique black arrows). In contrast to many other types of large neurons, the Purkinje cell bodies contain only small amounts of glycogen (white arrows). Magnification: 200x, same magnification as in panel A . b′. Glycogen accumulation is prominent also in the molecular layer and in the cell bodies (horizontal black arrows) and apical cytoplasmic processes (Bergman fibers, oblique black arrows) of Golgi epithelial cell astrocytes, but not in basket or stellate neurons. Magnification: 200x; bar = 20 μm.
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    Millipore filipin complex
    The fluorescent images of PAMAM–PEG–SRL/ EMA-labeled DNA cellular internalization. Internalization mechanism of nanoparticles by C6 glioma cells. Concentration of PAMAM of all samples was adjusted to 1μM (250 μg/m). PAMAM–PEG–SRL/EMA-labeled DNA without any inhibitor (as control) (A, B). Cells were incubated with different endocytosis inhibitors including: lactoferrin (E, F), phenylarsine oxide (G, H), <t>filipin</t> complex (I, J), colchicine (K, L), and at 4 o C (C, D); for 30 min. Red: EMA-labeled DNA. Bar: 50μm
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    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: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore prepared filipin complex
    ( See previous page ). MYO1C-depleted cells contain more total cellular cholesterol, trapped in intracellular storage compartments. ( A ) Confocal z-projection microscopy images of cellular cholesterol, imaged with <t>filipin,</t> from mock and MYO1C siRNA-depleted HeLa cells. Scale bar = 20 μm ( B ) Total cholesterol levels in mock and MYO1C-knockdown cells and DMSO- and PClP-treated cells were quantified by high-throughput microscopy. Automated imaging and analysis software was used to calculate the total filipin fluorescence per cell. Loss of MYO1C caused a significant increase in cholesterol as compared to control cells. In total > 149 ,000 cells from 3 independent experiments, each performed in triplicate, were analyzed. Graphs represent the means ± s.e.m. ( C ) Model of the autophagy and endocytic pathway highlighting defects (shown in blue) observed in MYO1C-depleted cells. Loss of functional MYO1C causes a defect in lipid raft recycling from the perinuclear recycling compartment back to the cell surface. This leads to intracellular accumulation of cholesterol-enriched membranes. 6 In the classical endocytic pathway, incoming cargo first moves through early endosomes, which then acquire an increasing number of intralumenal vesicles and mature into late endosomes (LE)/multivesicular bodies (MVB). The fusion of a LE/MVB with a lysosome generates a transient hybrid organelle, the endolysosome, in which content degradation can take place. In MYO1C-depleted cells, we observe the accumulation of enlarged LAMP1- and CTSD-positive endolysosomes. In the autophagy pathway, cytosolic material is sequestered by expansion and closure of a phagophore, forming double-membrane vesicles called autophagosomes. These autophagosomes mature by fusion with early and late endosomes to form an intermediate organelle called the amphisome, which fuses with lysosomes to enable content degradation. Ablating MYO1C activity leads to an accumulation in the number of autophagic structures suggesting a block in fusion of autophagic organelles with lysosomes.
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    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: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    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: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    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
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    Millipore filipin sigma stock solution
    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 Sigma Stock Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Millipore filipine
    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
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    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.
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    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.
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    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.
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    Millipore lipid raft lr 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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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
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    Image Search Results


    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

    Effect of methyl-β-cyclodextrin (MβCD) on reduction of cholesterol accumulation and lysosome size in NPC fibroblasts determined by Lysotracker-red staining, filipin cholesterol staining and Amplex-red cholesterol assays. (A) Images of

    Journal: Journal of biomolecular screening

    Article Title: A Phenotypic Compound Screening Assay for Lysosomal Storage Diseases

    doi: 10.1177/1087057113501197

    Figure Lengend Snippet: Effect of methyl-β-cyclodextrin (MβCD) on reduction of cholesterol accumulation and lysosome size in NPC fibroblasts determined by Lysotracker-red staining, filipin cholesterol staining and Amplex-red cholesterol assays. (A) Images of

    Article Snippet: Filipin dye (# F9765) and methyl-beta-cyclodextrin (#M7439) were obtained from Sigma-Aldrich.

    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

    MβCD increases autophagosome formation and restores impaired autophagy flux in NPC1 cells. (A) Autophagy detection in both WT and NPC1 fibroblasts, NSCs and neurons. Cells were treated with the indicated compounds (100 µM MβCD, 100 nM bafilomycin A 1 (baf. A1) or MβCD plus baf. A1) for 24 h, followed by western blot analysis. For this and all subsequent figures, a representative western blot is shown, and the bar graph represents the mean ± SEM of at least 3 replicates, unless otherwise noted. The intensity of LC3B-II and SQSTM1 were normalized with GAPDH. (B) Time-courses of LC3B and SQSTM1 levels in NPC1 cells after treatment with 100 µM MβCD. Cells were treated for the indicated times and analyzed by western blot. LC3B-II and SQSTM1 expression were normalized with GAPDH expression. (C) Quantification of LC3B and SQSTM1 immunofluorescence in WT and NPC1 fibroblasts and NSCs displayed in Fig. S2A. The normalized relative fluorescence intensities of LC3B and SQSTM1 dots are shown in bar graphs (mean ± SEM for at least 3 fields). (D) Time courses for effect of MβCD on reduction of cholesterol accumulation in NPC1 fibroblasts, NSCs and neurons. NPC1 cells were cultured in 96-well plates and treated with 100 µM MβCD for the indicated days, followed by the filipin staining assay. Data represent mean ± SEM of 3 replicates. For this and all subsequent figures, * p

    Journal: Autophagy

    Article Title: Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

    doi: 10.1080/15548627.2017.1329081

    Figure Lengend Snippet: MβCD increases autophagosome formation and restores impaired autophagy flux in NPC1 cells. (A) Autophagy detection in both WT and NPC1 fibroblasts, NSCs and neurons. Cells were treated with the indicated compounds (100 µM MβCD, 100 nM bafilomycin A 1 (baf. A1) or MβCD plus baf. A1) for 24 h, followed by western blot analysis. For this and all subsequent figures, a representative western blot is shown, and the bar graph represents the mean ± SEM of at least 3 replicates, unless otherwise noted. The intensity of LC3B-II and SQSTM1 were normalized with GAPDH. (B) Time-courses of LC3B and SQSTM1 levels in NPC1 cells after treatment with 100 µM MβCD. Cells were treated for the indicated times and analyzed by western blot. LC3B-II and SQSTM1 expression were normalized with GAPDH expression. (C) Quantification of LC3B and SQSTM1 immunofluorescence in WT and NPC1 fibroblasts and NSCs displayed in Fig. S2A. The normalized relative fluorescence intensities of LC3B and SQSTM1 dots are shown in bar graphs (mean ± SEM for at least 3 fields). (D) Time courses for effect of MβCD on reduction of cholesterol accumulation in NPC1 fibroblasts, NSCs and neurons. NPC1 cells were cultured in 96-well plates and treated with 100 µM MβCD for the indicated days, followed by the filipin staining assay. Data represent mean ± SEM of 3 replicates. For this and all subsequent figures, * p

    Article Snippet: Filipin staining Filipin dye (Sigma-Aldrich, F9765) detects the unesterified cholesterol in cells.

    Techniques: Western Blot, Expressing, Immunofluorescence, Fluorescence, Cell Culture, Staining

    PRKAB1 or PRKAB2 is required for the effect of MβCD on cholesterol reduction, autophagy induction, and increase of autophagy flux. (A, B) Filipin staining of NPC1 fibroblasts treated with MβCD. (A) PRKAB1 or PRKAB2 expression was silenced by shRNA and the subunits re-expressed with transient transfection of either PRKAB1 or PRKAB2 activation vectors. (B) ATG12 expression was silenced by shRNA. All of the cells were treated with 100 µM MβCD or DMSO control for 4 d followed by the filipin staining assay. (C) NPC1 fibroblasts transfected with ATG12 shRNA or control shRNA were treated with 100 µM MβCD or DMSO control for 24 h, followed by western blot analysis with the indicated antibodies. LC3B-II, SQSTM1 and ATG12 expression were normalized to GAPDH expression. (D-G) MβCD effects on SNARE proteins interactions. (D) Immunoprecipitation and western blot analysis of 3 SNARE proteins (VAMP8, STX17 and SNAP29). WT fibroblasts with the PRKAB/AMPK β-subunit silenced or with AMPK β-subunit re-expression was treated with MβCD or DMSO for 24 h. Cells were lysed and directly immunoprecipitated with anti-VAMP8 antibody followed by western blot analysis with the indicated antibodies. (E) NPC1 and WT fibroblasts were treated with MβCD for the indicated times, followed by immunoprecipitation with anti-SNAP29 and western blot analysis with the indicated antibodies. (F) Immunofluorescence staining and colocalization of LC3 with VAMP8. Indicated WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles, were treated with 100 µM MβCD or DMSO for 24 h and stained with anti-VAMP8 antibody. The punctate structures of VAMP8 were colocalized with RFP-LC3 (yellow color in the merged images). Data represent mean ± SEM of 10 images. (G) NPC1 and WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles were treated with MβCD for the indicated times, followed by staining with anti-VAMP8 antibody. The colocalization of VAMP8 and RFP-LC3 puncta was analyzed as above. Abbreviations: PRKAB1 or PRKAB2 act., PRKAB1 or PRKAB2 activation vectors; Mock vec., mock vector; LY, lysosome; AP, autophagosome. Scale bar: 10 µm (yellow) and 1 µm (white).

    Journal: Autophagy

    Article Title: Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

    doi: 10.1080/15548627.2017.1329081

    Figure Lengend Snippet: PRKAB1 or PRKAB2 is required for the effect of MβCD on cholesterol reduction, autophagy induction, and increase of autophagy flux. (A, B) Filipin staining of NPC1 fibroblasts treated with MβCD. (A) PRKAB1 or PRKAB2 expression was silenced by shRNA and the subunits re-expressed with transient transfection of either PRKAB1 or PRKAB2 activation vectors. (B) ATG12 expression was silenced by shRNA. All of the cells were treated with 100 µM MβCD or DMSO control for 4 d followed by the filipin staining assay. (C) NPC1 fibroblasts transfected with ATG12 shRNA or control shRNA were treated with 100 µM MβCD or DMSO control for 24 h, followed by western blot analysis with the indicated antibodies. LC3B-II, SQSTM1 and ATG12 expression were normalized to GAPDH expression. (D-G) MβCD effects on SNARE proteins interactions. (D) Immunoprecipitation and western blot analysis of 3 SNARE proteins (VAMP8, STX17 and SNAP29). WT fibroblasts with the PRKAB/AMPK β-subunit silenced or with AMPK β-subunit re-expression was treated with MβCD or DMSO for 24 h. Cells were lysed and directly immunoprecipitated with anti-VAMP8 antibody followed by western blot analysis with the indicated antibodies. (E) NPC1 and WT fibroblasts were treated with MβCD for the indicated times, followed by immunoprecipitation with anti-SNAP29 and western blot analysis with the indicated antibodies. (F) Immunofluorescence staining and colocalization of LC3 with VAMP8. Indicated WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles, were treated with 100 µM MβCD or DMSO for 24 h and stained with anti-VAMP8 antibody. The punctate structures of VAMP8 were colocalized with RFP-LC3 (yellow color in the merged images). Data represent mean ± SEM of 10 images. (G) NPC1 and WT fibroblasts, transiently transfected with TagRFP-LC3 lentiviral particles were treated with MβCD for the indicated times, followed by staining with anti-VAMP8 antibody. The colocalization of VAMP8 and RFP-LC3 puncta was analyzed as above. Abbreviations: PRKAB1 or PRKAB2 act., PRKAB1 or PRKAB2 activation vectors; Mock vec., mock vector; LY, lysosome; AP, autophagosome. Scale bar: 10 µm (yellow) and 1 µm (white).

    Article Snippet: Filipin staining Filipin dye (Sigma-Aldrich, F9765) detects the unesterified cholesterol in cells.

    Techniques: Staining, Expressing, shRNA, Transfection, Activation Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Activated Clotting Time Assay, Plasmid Preparation

    MβCD binds to PRKAB1 and PRKAB2 with a higher affinity for PRKAB1. (A) Temperature melting curves of PRKAB1 and PRKAB2 in the presence or absence of 300 µM MβCD. The relative chemiluminescent intensity of each sample at different temperatures was used to generate temperature-dependent melting curves and the apparent aggregation temperature (T agg ) was calculated by nonlinear regression. (B) Apparent binding affinities of MβCD with PRKAB1 and PRKAB2 measured by CETSA. Cell lysates were treated with MβCD and heated to 53°C for 3 min. The supernatants obtained after centrifugation were analyzed by western blotting using anti-PRKAB1 or anti-PRKAB2 antibody. A representative blot was shown and data represent mean ± SEM of at least 3 replicates. (C) MβCD reduced NPC1 phenotypes in fibroblasts, NSCs and neurons. NPC1 cells were cultured in 96-well plates and treated with various concentrations of MβCD. After a 4-d (fibroblasts and NSCs) or 3-d (neurons) incubation filipin or LysoTracker Red staining was performed. Data represent mean ± SEM of 3 replicates. (D) A structural model of AMPK (PRKAA2B1G1, PDB code 4CFF) bound with MβCD (yellow), activator A769662 (brown), and inhibitor dorsomorphin (gray). The 3 subunits of AMPK are shown in green (PRKA2), magenta (PRKAB1), and cyan (PRKAG1). (E) Binding model of MβCD with PRKAB1. (F) Binding model of MβCD with PRKAB2. MβCD is shown in sticks in yellow (carbon atom). AMPK is shown in ribbons and key interacting residues are shown in sticks in green (PRKAB1) or cyan (PRKAB2). Residue L146 within a flexible loop, which is positioned in the MβCD hole, is shown in magenta.

    Journal: Autophagy

    Article Title: Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

    doi: 10.1080/15548627.2017.1329081

    Figure Lengend Snippet: MβCD binds to PRKAB1 and PRKAB2 with a higher affinity for PRKAB1. (A) Temperature melting curves of PRKAB1 and PRKAB2 in the presence or absence of 300 µM MβCD. The relative chemiluminescent intensity of each sample at different temperatures was used to generate temperature-dependent melting curves and the apparent aggregation temperature (T agg ) was calculated by nonlinear regression. (B) Apparent binding affinities of MβCD with PRKAB1 and PRKAB2 measured by CETSA. Cell lysates were treated with MβCD and heated to 53°C for 3 min. The supernatants obtained after centrifugation were analyzed by western blotting using anti-PRKAB1 or anti-PRKAB2 antibody. A representative blot was shown and data represent mean ± SEM of at least 3 replicates. (C) MβCD reduced NPC1 phenotypes in fibroblasts, NSCs and neurons. NPC1 cells were cultured in 96-well plates and treated with various concentrations of MβCD. After a 4-d (fibroblasts and NSCs) or 3-d (neurons) incubation filipin or LysoTracker Red staining was performed. Data represent mean ± SEM of 3 replicates. (D) A structural model of AMPK (PRKAA2B1G1, PDB code 4CFF) bound with MβCD (yellow), activator A769662 (brown), and inhibitor dorsomorphin (gray). The 3 subunits of AMPK are shown in green (PRKA2), magenta (PRKAB1), and cyan (PRKAG1). (E) Binding model of MβCD with PRKAB1. (F) Binding model of MβCD with PRKAB2. MβCD is shown in sticks in yellow (carbon atom). AMPK is shown in ribbons and key interacting residues are shown in sticks in green (PRKAB1) or cyan (PRKAB2). Residue L146 within a flexible loop, which is positioned in the MβCD hole, is shown in magenta.

    Article Snippet: Filipin staining Filipin dye (Sigma-Aldrich, F9765) detects the unesterified cholesterol in cells.

    Techniques: Binding Assay, Centrifugation, Western Blot, Cell Culture, Incubation, Staining

    Pharmacological effect of MβCD is blocked by an inhibitor and mimicked by activators of AMPK. (A, B) Filipin staining for unesterified cholesterol accumulation in WT and NPC1 fibroblasts treated with the AMPK inhibitor dorsomorphin (DM) in the presence and absence of 100 µM MβCD. (C) Western blotting for LC3B and SQSTM1 levels in NPC1 fibroblasts treated with the indicated compounds (2 µM DM, 100 µM MβCD, or 100 nM baf. A1) for 24 h. LC3B-II and SQSTM1 expression were normalized to GAPDH expression. (D) Filipin staining after the cells were treated with the indicated compounds (1.25 µM RSVA 405, 200 µM A769662 or 100 nM baf. A1) for 4 d. (E) Western blotting for LC3B and SQSTM1 levels in NPC1 fibroblasts treated with AMPK activators (1.25 µM RSVA 405 or 200 µM A769662) for 24 h. LC3-II and SQSTM1 expression were normalized to GAPDH expression. Scale bar: 10 µm.

    Journal: Autophagy

    Article Title: Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

    doi: 10.1080/15548627.2017.1329081

    Figure Lengend Snippet: Pharmacological effect of MβCD is blocked by an inhibitor and mimicked by activators of AMPK. (A, B) Filipin staining for unesterified cholesterol accumulation in WT and NPC1 fibroblasts treated with the AMPK inhibitor dorsomorphin (DM) in the presence and absence of 100 µM MβCD. (C) Western blotting for LC3B and SQSTM1 levels in NPC1 fibroblasts treated with the indicated compounds (2 µM DM, 100 µM MβCD, or 100 nM baf. A1) for 24 h. LC3B-II and SQSTM1 expression were normalized to GAPDH expression. (D) Filipin staining after the cells were treated with the indicated compounds (1.25 µM RSVA 405, 200 µM A769662 or 100 nM baf. A1) for 4 d. (E) Western blotting for LC3B and SQSTM1 levels in NPC1 fibroblasts treated with AMPK activators (1.25 µM RSVA 405 or 200 µM A769662) for 24 h. LC3-II and SQSTM1 expression were normalized to GAPDH expression. Scale bar: 10 µm.

    Article Snippet: Filipin staining Filipin dye (Sigma-Aldrich, F9765) detects the unesterified cholesterol in cells.

    Techniques: Staining, Western Blot, Expressing

    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

    Itraconazole's effects on VEGFR2 glycosylation occur in parallel to other itraconazole-induced effects. A , cholesterol localization was visualized by filipin staining of HUVEC treated with 800 n m itraconazole ( Ita ), 500 μ m castanospermine ( Cast

    Journal: The Journal of Biological Chemistry

    Article Title: The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells *

    doi: 10.1074/jbc.M111.278754

    Figure Lengend Snippet: Itraconazole's effects on VEGFR2 glycosylation occur in parallel to other itraconazole-induced effects. A , cholesterol localization was visualized by filipin staining of HUVEC treated with 800 n m itraconazole ( Ita ), 500 μ m castanospermine ( Cast

    Article Snippet: 200 μl of filipin staining solution (50 μg/ml filipin (Sigma, catalog no. F9765) diluted from a 100× stock in DSMO stored in the dark and made from powder stored under argon) and slides were incubated in the dark for 2 h at room temperature.

    Techniques: Staining

    Effects of different sources of MβCDs on reducing cholesterol accumulation in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) and WT control (GM05659) were untreated or treated with MβCD (0.4–300 μM) for 4 days; filipin staining was then performed. (A) Images of filipin staining on NPC1 fibroblasts. Treatment with 300, 11 μM of MβCD-3 significantly reduced cholesterol accumulation in NPC1 patient skin fibroblasts, while the other two batches of MβCDs (MβCD-1 and MβCD-2) showed much weaker effects on cholesterol accumulation in NPC1 patient fibroblasts. Filipin ( green ) stains the intracellular cholesterol-laden domains, and EthD-1 ( red ) stains nuclei. (B) Treatment with MβCD-3 (300 μM) significantly reduced cholesterol accumulation in the NPC1 patient fibroblast compared with the other two batches of MβCDs (MβCD-1 and MβCD-2). (C) Dose–response curve of different sources of MβCDs on cholesterol accumulation in NPC1 patient fibroblasts. EthD-1, ethidium homodimer; NPC1, Niemann-Pick disease type C1; WT, wild-type.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Effects of different sources of MβCDs on reducing cholesterol accumulation in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) and WT control (GM05659) were untreated or treated with MβCD (0.4–300 μM) for 4 days; filipin staining was then performed. (A) Images of filipin staining on NPC1 fibroblasts. Treatment with 300, 11 μM of MβCD-3 significantly reduced cholesterol accumulation in NPC1 patient skin fibroblasts, while the other two batches of MβCDs (MβCD-1 and MβCD-2) showed much weaker effects on cholesterol accumulation in NPC1 patient fibroblasts. Filipin ( green ) stains the intracellular cholesterol-laden domains, and EthD-1 ( red ) stains nuclei. (B) Treatment with MβCD-3 (300 μM) significantly reduced cholesterol accumulation in the NPC1 patient fibroblast compared with the other two batches of MβCDs (MβCD-1 and MβCD-2). (C) Dose–response curve of different sources of MβCDs on cholesterol accumulation in NPC1 patient fibroblasts. EthD-1, ethidium homodimer; NPC1, Niemann-Pick disease type C1; WT, wild-type.

    Article Snippet: Filipin Fluorescence Staining of Free Cholesterol Filipin dye (catalog No. F9765; Sigma) is a polyene macrolide antibiotic and antifungal compound.

    Techniques: Staining, Ethidium Homodimer Assay

    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

    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

    Cerebellar cortex, 15-month-old knockout mice. ( A ) Glycol methacrylate-embedded tissue sectioned at 3 μm, stained with Richardson’s stain. Purkinje neuron cell bodies (white arrows) are mildly vacuolated (and almost free of glycogen, as seen in B ), whereas the cell bodies of adjacent Golgi epithelial cells (radially-oriented astrocytes) are markedly vacuolated. Magnification: 200x; bar: 20 μm. a′. Cryostat section, 20-μm-thick stained with the fluorescent dye, filipin, shows intense accumulation of cholesterol in the apical cytoplasm of Purkinje neurons (white arrows), even though these cells differ from most other large neurons in not accumulating comparable amounts of glycogen ( B ). Magnification: 100x. ( B ) Paraffin section, 7 μm, stained with the PAS method and counterstained with hematoxylin. There is marked glycogen accumulation in the cell bodies of Golgi epithelial cell astrocytes (horizontal black arrows) and in clumps in the granular layer and in the cytoplasm of most glial cells in the cerebellar cortical white matter (oblique black arrows). In contrast to many other types of large neurons, the Purkinje cell bodies contain only small amounts of glycogen (white arrows). Magnification: 200x, same magnification as in panel A . b′. Glycogen accumulation is prominent also in the molecular layer and in the cell bodies (horizontal black arrows) and apical cytoplasmic processes (Bergman fibers, oblique black arrows) of Golgi epithelial cell astrocytes, but not in basket or stellate neurons. Magnification: 200x; bar = 20 μm.

    Journal: Journal of neuropathology and experimental neurology

    Article Title: Temporal Neuropathological and Behavioral Phenotype of 6Neo/6Neo Pompe Disease Mice

    doi: 10.1097/NEN.0b013e3181815994

    Figure Lengend Snippet: Cerebellar cortex, 15-month-old knockout mice. ( A ) Glycol methacrylate-embedded tissue sectioned at 3 μm, stained with Richardson’s stain. Purkinje neuron cell bodies (white arrows) are mildly vacuolated (and almost free of glycogen, as seen in B ), whereas the cell bodies of adjacent Golgi epithelial cells (radially-oriented astrocytes) are markedly vacuolated. Magnification: 200x; bar: 20 μm. a′. Cryostat section, 20-μm-thick stained with the fluorescent dye, filipin, shows intense accumulation of cholesterol in the apical cytoplasm of Purkinje neurons (white arrows), even though these cells differ from most other large neurons in not accumulating comparable amounts of glycogen ( B ). Magnification: 100x. ( B ) Paraffin section, 7 μm, stained with the PAS method and counterstained with hematoxylin. There is marked glycogen accumulation in the cell bodies of Golgi epithelial cell astrocytes (horizontal black arrows) and in clumps in the granular layer and in the cytoplasm of most glial cells in the cerebellar cortical white matter (oblique black arrows). In contrast to many other types of large neurons, the Purkinje cell bodies contain only small amounts of glycogen (white arrows). Magnification: 200x, same magnification as in panel A . b′. Glycogen accumulation is prominent also in the molecular layer and in the cell bodies (horizontal black arrows) and apical cytoplasmic processes (Bergman fibers, oblique black arrows) of Golgi epithelial cell astrocytes, but not in basket or stellate neurons. Magnification: 200x; bar = 20 μm.

    Article Snippet: Cryostat sections cut at 20 μm were stained for cholesterol with the filipin reagent (Sigma-Aldrich), 10 mg/ml in PBS for 3 hours, and were examined by fluorescence microscopy.

    Techniques: Knock-Out, Mouse Assay, Staining, Paraffin Section

    Paraffin section, 7 μm, PAS-hematoxylin. The hippocampal formation (dentate gyrus, hippocampus CA1, Ca2, and CA3, and subiculum) occupies the center of the field, and a sector of cerebral neocortex lies in the left part of the field of a 15-month-old knockout mouse. Glial cells contain increased glycogen in the radiatum and lacunosum layers of the hippocampus, in the dentate gyrus and subiculum, in the corpus callosum dorsal to the hippocampus, while glycogen-rich glial and vascular cells lie in the gray matter of the cerebral cortex (top left) and thalamus (left of inset). The inset shows lysosomal fluorescence (white), due to binding of the filipin reagent to lysosomal cholesterol in the apical cytoplasm of CA1 pyramidal neurons, even though these cells, delimited by the black bars in the colored part of the figure, are predominantly glycogen-free. Bar: 500 μm.

    Journal: Journal of neuropathology and experimental neurology

    Article Title: Temporal Neuropathological and Behavioral Phenotype of 6Neo/6Neo Pompe Disease Mice

    doi: 10.1097/NEN.0b013e3181815994

    Figure Lengend Snippet: Paraffin section, 7 μm, PAS-hematoxylin. The hippocampal formation (dentate gyrus, hippocampus CA1, Ca2, and CA3, and subiculum) occupies the center of the field, and a sector of cerebral neocortex lies in the left part of the field of a 15-month-old knockout mouse. Glial cells contain increased glycogen in the radiatum and lacunosum layers of the hippocampus, in the dentate gyrus and subiculum, in the corpus callosum dorsal to the hippocampus, while glycogen-rich glial and vascular cells lie in the gray matter of the cerebral cortex (top left) and thalamus (left of inset). The inset shows lysosomal fluorescence (white), due to binding of the filipin reagent to lysosomal cholesterol in the apical cytoplasm of CA1 pyramidal neurons, even though these cells, delimited by the black bars in the colored part of the figure, are predominantly glycogen-free. Bar: 500 μm.

    Article Snippet: Cryostat sections cut at 20 μm were stained for cholesterol with the filipin reagent (Sigma-Aldrich), 10 mg/ml in PBS for 3 hours, and were examined by fluorescence microscopy.

    Techniques: Paraffin Section, Knock-Out, Fluorescence, Binding Assay

    VDR silencing (shVDR) in HepG2 cells demonstrates that VDR is a potential negative regulator of FETUB. A. Representative PCR performed on gDNA from shVDR clones, viral controls (EB8 and EB10) and HepG2 cells (-ve control) using backbone specific primers to demonstrate successful integration. Concentration and gDNA quality were assessed a Gapdh PCR. Integration PCRs were performed for each independent shRNA delivery (n = 3). B. Western immunoblot analyses under reduced conditions in 20ug of whole cell lysates from shVDR clones (shVDR) and controls (CTR). Antibodies specific for VDR, FETUB, RXRα and ERK were utilized to analyze their endogenous expression, with results normalized to total protein. Gels are representative of n = 3 experiments for 3 independent viral deliveries performed. C. Representative western immunoblot analyses for RXRα and ERK of 2DE with 100ug of whole cell lysates from shVDR clones (shVDR542) and controls (EB8). Arrows highlight pI shifts observed for RXRα and ERK1 p44 and ERK2 p42. D. Representative western immunoblot analyses of endogenous FETUB (FETUBendog) under reduced conditions in 20ug of whole cell lysates from shVDR clones and controls (shEB8 or HepG2) following a 1hr treatment with increasing concentrations of filipin (1–10 ug/ml) or CPZH (5-10ug/ml), followed by 24 h in maintenance medium. Results are representative of 4 independent shVDR clones and are normalized to total protein (n = 3).

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

    Article Title: Fetuin B links vitamin D deficiency and pediatric obesity: Direct negative regulation by vitamin D

    doi: 10.1016/j.jsbmb.2018.04.009

    Figure Lengend Snippet: VDR silencing (shVDR) in HepG2 cells demonstrates that VDR is a potential negative regulator of FETUB. A. Representative PCR performed on gDNA from shVDR clones, viral controls (EB8 and EB10) and HepG2 cells (-ve control) using backbone specific primers to demonstrate successful integration. Concentration and gDNA quality were assessed a Gapdh PCR. Integration PCRs were performed for each independent shRNA delivery (n = 3). B. Western immunoblot analyses under reduced conditions in 20ug of whole cell lysates from shVDR clones (shVDR) and controls (CTR). Antibodies specific for VDR, FETUB, RXRα and ERK were utilized to analyze their endogenous expression, with results normalized to total protein. Gels are representative of n = 3 experiments for 3 independent viral deliveries performed. C. Representative western immunoblot analyses for RXRα and ERK of 2DE with 100ug of whole cell lysates from shVDR clones (shVDR542) and controls (EB8). Arrows highlight pI shifts observed for RXRα and ERK1 p44 and ERK2 p42. D. Representative western immunoblot analyses of endogenous FETUB (FETUBendog) under reduced conditions in 20ug of whole cell lysates from shVDR clones and controls (shEB8 or HepG2) following a 1hr treatment with increasing concentrations of filipin (1–10 ug/ml) or CPZH (5-10ug/ml), followed by 24 h in maintenance medium. Results are representative of 4 independent shVDR clones and are normalized to total protein (n = 3).

    Article Snippet: At 60% confluency, they were treated dose dependently with 1–10 ug/ml of Filipin (Sigma), or 5–10 ug/ml of chlorpromazine hydrochloride (CPZH) with the appropriate excipient for 1hr in serum free medium to block endocytosis.

    Techniques: Polymerase Chain Reaction, Clone Assay, Concentration Assay, shRNA, Western Blot, Expressing, Two-Dimensional Gel Electrophoresis

    IFITM3 and 2 induce accumulation of cholesterol in endosomal compartments. (A). Analysis of cholesterol distribution using the specific marker filipin (red) in Vero-IFITM1, 2 and 3 cells (green) and cells containing the empty vector. Cholesterol is accumulated in endosomes upon expression of IFITM2 and 3. (B). Basal IFITM2 expression in control cells containing the empty vector did not produce cholesterol accumulation. N = 30 cells per condition. Bar = 10μm.

    Journal: PLoS ONE

    Article Title: Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection

    doi: 10.1371/journal.pone.0154366

    Figure Lengend Snippet: IFITM3 and 2 induce accumulation of cholesterol in endosomal compartments. (A). Analysis of cholesterol distribution using the specific marker filipin (red) in Vero-IFITM1, 2 and 3 cells (green) and cells containing the empty vector. Cholesterol is accumulated in endosomes upon expression of IFITM2 and 3. (B). Basal IFITM2 expression in control cells containing the empty vector did not produce cholesterol accumulation. N = 30 cells per condition. Bar = 10μm.

    Article Snippet: To detect cholesterol distribution we used Filipin staining (Sigma), as previously described [ ].

    Techniques: Marker, Plasmid Preparation, Expressing

    Inhibition of cholesterol transport occurs with JNJ-26854165 treatment. (A) WT MEFs and JNJ-26854165-resistant MEFs (165R) were visualized by light microscopy (40× magnification). (B) Fluorescent staining of cholesterol using Filipin in WT and

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.113.204958

    Figure Lengend Snippet: Inhibition of cholesterol transport occurs with JNJ-26854165 treatment. (A) WT MEFs and JNJ-26854165-resistant MEFs (165R) were visualized by light microscopy (40× magnification). (B) Fluorescent staining of cholesterol using Filipin in WT and

    Article Snippet: Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich).

    Techniques: Inhibition, Light Microscopy, Staining

    Decreased cholesterol efflux and transport induced by JNJ-26854165 in MM and MCL cells. (A) MM and MCL cell lines were treated with 5 μ M/l JNJ-26854165 for 24 hours stained with Filipin and cholesterol localization determined using an ImageStream

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.113.204958

    Figure Lengend Snippet: Decreased cholesterol efflux and transport induced by JNJ-26854165 in MM and MCL cells. (A) MM and MCL cell lines were treated with 5 μ M/l JNJ-26854165 for 24 hours stained with Filipin and cholesterol localization determined using an ImageStream

    Article Snippet: Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich).

    Techniques: Staining

    Lovastatin induced cell apoptosis, and sensitized cancer cells to TRAIL-induced apoptosis A. Representative cells to enable visualization and overlays of lipid rafts/cholesterol in non-malignant and cancer cell lines, using confocal microscopy. Non-malignant cells PZ-HPV-7 and MCF10A and prostate cancer cells C4-2, PC-3 and LNCaP were labeled with Alexa red Fluo555/565 – CTXB (lipid rafts, glycolipoprotein microdomains, GM) and green filipin (cholesterol, CH) and analyzed by confocal microscopy. B. The correlation of level of lipid rafts/cholesterol and the sensitivity of cells to lovastatin-induced apoptosis. Serum-starved cells were treated with lovastatin at 10 μM or DSMO control for 16 hours, with triplicate-wells for each cell line. Apoptotic cells were stained with Annexin V-FITC and PI, and detected by flow cytometry. Data were expressed as the ratio of lovastatin-treated cells to control. C. The effect of lovastatin on the level of cholesterol in the lipid rafts and apoptosis in prostate cancer cells. Serum-starved cells were treated with DSMO (control) or lovastatin at 10 μM for 16 hours, and then incubated with or without 500 μM cholesterol for 2 hours. Cells were stained with CTXB-Alexa 555/558 (GM) and filipin (CH), and monitored by confocal microscopy. The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry. Each experiment was replicated 3 times. D. Prostate cancer cells are resistant to TRAIL-induced apoptosis. Prostate cancer cells LNCap, C4-2, CWR22rv, PC-3, DU145, non-malignant prostate epithelial cells PZ-HPV-7, keratinocytes, non small lung adenocarcinoma cells A549 and colon cancer Lovo cells were treated with TRAIL protein for 24 hours at a range of doses (n=5 for each cell line). Cell viability was measured by MTT assay at 72 hours after drug treatment. E. Lovastatin significantly enhanced TRAIL-induced apoptosis in prostate cancer cells, but not in normal cells. LNCaP, C4-2, PC-3, PZ-HPV-7 and keratinocytes were treated with lovastatin at 10 μM for 16 hours, before being treated with or without TRAIL(200 ng/mL) for 24 hours (n=4/group). The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry.

    Journal: Oncotarget

    Article Title: Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells

    doi:

    Figure Lengend Snippet: Lovastatin induced cell apoptosis, and sensitized cancer cells to TRAIL-induced apoptosis A. Representative cells to enable visualization and overlays of lipid rafts/cholesterol in non-malignant and cancer cell lines, using confocal microscopy. Non-malignant cells PZ-HPV-7 and MCF10A and prostate cancer cells C4-2, PC-3 and LNCaP were labeled with Alexa red Fluo555/565 – CTXB (lipid rafts, glycolipoprotein microdomains, GM) and green filipin (cholesterol, CH) and analyzed by confocal microscopy. B. The correlation of level of lipid rafts/cholesterol and the sensitivity of cells to lovastatin-induced apoptosis. Serum-starved cells were treated with lovastatin at 10 μM or DSMO control for 16 hours, with triplicate-wells for each cell line. Apoptotic cells were stained with Annexin V-FITC and PI, and detected by flow cytometry. Data were expressed as the ratio of lovastatin-treated cells to control. C. The effect of lovastatin on the level of cholesterol in the lipid rafts and apoptosis in prostate cancer cells. Serum-starved cells were treated with DSMO (control) or lovastatin at 10 μM for 16 hours, and then incubated with or without 500 μM cholesterol for 2 hours. Cells were stained with CTXB-Alexa 555/558 (GM) and filipin (CH), and monitored by confocal microscopy. The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry. Each experiment was replicated 3 times. D. Prostate cancer cells are resistant to TRAIL-induced apoptosis. Prostate cancer cells LNCap, C4-2, CWR22rv, PC-3, DU145, non-malignant prostate epithelial cells PZ-HPV-7, keratinocytes, non small lung adenocarcinoma cells A549 and colon cancer Lovo cells were treated with TRAIL protein for 24 hours at a range of doses (n=5 for each cell line). Cell viability was measured by MTT assay at 72 hours after drug treatment. E. Lovastatin significantly enhanced TRAIL-induced apoptosis in prostate cancer cells, but not in normal cells. LNCaP, C4-2, PC-3, PZ-HPV-7 and keratinocytes were treated with lovastatin at 10 μM for 16 hours, before being treated with or without TRAIL(200 ng/mL) for 24 hours (n=4/group). The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry.

    Article Snippet: After rinsing in PBS, the cells were incubated with 1 ml of Filipin working solution (0.05 mg/ml in PBS, Sigma Aldrich, St. Louis, MO, USA) for 2 h at room temperature.

    Techniques: Confocal Microscopy, Labeling, Staining, Flow Cytometry, Cytometry, Incubation, MTT Assay

    Two alternative routes on filipin III biosynthesis in S. filipinensis.

    Journal: Microbial Cell Factories

    Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

    doi: 10.1186/s12934-015-0307-4

    Figure Lengend Snippet: Two alternative routes on filipin III biosynthesis in S. filipinensis.

    Article Snippet: Minimum inhibitory concentrations (MICs) were determined by the broth microdilution technique following the EUCAST methodology by diluting filipin III derivatives in RPMI 1640 with glutamine and 0.2% glucose but without sodium bicarbonate (Sigma) buffered with 0.164 M MOPS pH 7.0 to concentrations of 40 μg/ml of which 100 μl was added to the first row of a 96-well suspension culture plate.

    Techniques:

    Bioassay and MICs of filipin III and its intermediates. Bioassays were performed with ethyl acetate-extracted culture broths from early stationary phase-grown cells. C. utilis was used as test organism. MICs of the purified filipin derivatives for C. utilis and S. cerevisiae were measured by broth microdilution assay (see “ Methods ”). Abbreviations are as in Fig. 4 .

    Journal: Microbial Cell Factories

    Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

    doi: 10.1186/s12934-015-0307-4

    Figure Lengend Snippet: Bioassay and MICs of filipin III and its intermediates. Bioassays were performed with ethyl acetate-extracted culture broths from early stationary phase-grown cells. C. utilis was used as test organism. MICs of the purified filipin derivatives for C. utilis and S. cerevisiae were measured by broth microdilution assay (see “ Methods ”). Abbreviations are as in Fig. 4 .

    Article Snippet: Minimum inhibitory concentrations (MICs) were determined by the broth microdilution technique following the EUCAST methodology by diluting filipin III derivatives in RPMI 1640 with glutamine and 0.2% glucose but without sodium bicarbonate (Sigma) buffered with 0.164 M MOPS pH 7.0 to concentrations of 40 μg/ml of which 100 μl was added to the first row of a 96-well suspension culture plate.

    Techniques: Purification, Microdilution Assay

    FilI inactivation increases filipin III production. a Predicted PCR fragment amplification of the parental strain and the mutant. The primers used in the assay are indicated with arrowheads . The acc(3)IV-oriT cassette is indicated in black . b PCR analysis of the wild type and the mutant. c Growth curves and time course quantification of the filipin III production in S. filipinensis ( black ) and S. filipinensis ΔfilI ( grey ).

    Journal: Microbial Cell Factories

    Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

    doi: 10.1186/s12934-015-0307-4

    Figure Lengend Snippet: FilI inactivation increases filipin III production. a Predicted PCR fragment amplification of the parental strain and the mutant. The primers used in the assay are indicated with arrowheads . The acc(3)IV-oriT cassette is indicated in black . b PCR analysis of the wild type and the mutant. c Growth curves and time course quantification of the filipin III production in S. filipinensis ( black ) and S. filipinensis ΔfilI ( grey ).

    Article Snippet: Minimum inhibitory concentrations (MICs) were determined by the broth microdilution technique following the EUCAST methodology by diluting filipin III derivatives in RPMI 1640 with glutamine and 0.2% glucose but without sodium bicarbonate (Sigma) buffered with 0.164 M MOPS pH 7.0 to concentrations of 40 μg/ml of which 100 μl was added to the first row of a 96-well suspension culture plate.

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis

    Filipin intermediates generated upon gene inactivation. a TLC analyses of 72 h culture broth extracts of S. filipinensis ( 4 ), S. filipinensis ΔfilC ( 2 ), S. filipinensis ΔfilD ( 3 ), and S. filipinensis ΔfilCD ( 1 ). b Relative proportions of the major filipin products accumulated by the different strains. FI, filipin I (compound Y); FII, filipin II (compound Z); FIII, filipin III; 1´-OHFI, 1´-hydroxyfillipin I (compound X).

    Journal: Microbial Cell Factories

    Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

    doi: 10.1186/s12934-015-0307-4

    Figure Lengend Snippet: Filipin intermediates generated upon gene inactivation. a TLC analyses of 72 h culture broth extracts of S. filipinensis ( 4 ), S. filipinensis ΔfilC ( 2 ), S. filipinensis ΔfilD ( 3 ), and S. filipinensis ΔfilCD ( 1 ). b Relative proportions of the major filipin products accumulated by the different strains. FI, filipin I (compound Y); FII, filipin II (compound Z); FIII, filipin III; 1´-OHFI, 1´-hydroxyfillipin I (compound X).

    Article Snippet: Minimum inhibitory concentrations (MICs) were determined by the broth microdilution technique following the EUCAST methodology by diluting filipin III derivatives in RPMI 1640 with glutamine and 0.2% glucose but without sodium bicarbonate (Sigma) buffered with 0.164 M MOPS pH 7.0 to concentrations of 40 μg/ml of which 100 μl was added to the first row of a 96-well suspension culture plate.

    Techniques: Generated, Thin Layer Chromatography

    Fibroblast cell culture from the conjunctival biopsy of the proband. A : Fibroblastic cellular outgrowth from the conjunctival biopsy tissue. B : Following one week of cell culture, cellular confluence of fibroblasts is achieved. C : Following the addition of filipin stain, there is no evidence of cholesterol in the cultured conjunctival fibroblasts. D : Cells cultured with the addition of low density lipoprotein (LDL), acted as a positive control showing evidence of cholesterol with filipin staining. (Bar=100 microns).

    Journal: Molecular Vision

    Article Title: Analysis of conjunctival fibroblasts from a proband with Schnyder corneal dystrophy

    doi:

    Figure Lengend Snippet: Fibroblast cell culture from the conjunctival biopsy of the proband. A : Fibroblastic cellular outgrowth from the conjunctival biopsy tissue. B : Following one week of cell culture, cellular confluence of fibroblasts is achieved. C : Following the addition of filipin stain, there is no evidence of cholesterol in the cultured conjunctival fibroblasts. D : Cells cultured with the addition of low density lipoprotein (LDL), acted as a positive control showing evidence of cholesterol with filipin staining. (Bar=100 microns).

    Article Snippet: We then examined for non-esterified cholesterol using filipin stain (25µg/ml; a polyene antibiotic that selectively binds to the 3-beta-hydroxy-sterols; Sigma Aldrich) with a previously described method [ ].

    Techniques: Cell Culture, Staining, Positive Control

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0026289

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

    Article Snippet: Filipin staining Filipin stain (Sigma) was used to assess cholesterol distribution in the hair cell membrane in control and MβCD-treated cells .

    Techniques: Labeling, Staining

    Morphology and physiological features of cholesterol and SM-containing compartments. ( A ) TEM observations illustrating a representative field of the cytoplasm of NHF ( i ) and NPAF ( ii ). Note the abundance of membranous multilamellar inclusions in NPAFs (arrowheads and ii Inset ). m, mitochondria. ( B ) Fluorescence microscopy for analysis of lipid distribution. Staining of NHF ( i ) and NPAF ( ii ) with filipin and phase microscopy ( i′ and ii′ ) show cholesterol-laden organelles in NPAF. Cholesterol and sphingolipid colocalization is demonstrated by filipin ( iii ) and BODIPY-LacCer ( iv ) staining. The merged picture is presented in v (magnification: 100×). ( C ) Fluorescence microscopy ( i–iii ) and TEM ( iv ) showing the endocytic nature of the lipid-containing compartments. Incubation of NPAF with filipin ( i ) to identify cholesterol-containing organelles and with LysoTracker ( ii ) to track the endocytic structures resulted in a partial colocalization of the dyes (see merge in iii ). In iv , NPAF have been incubated with BSA-gold particles (15 nm) for 16 h at 37°C. Arrows highlight BSA-containing structures that morphologically resemble cholesterol and SM-containing compartments. (Scale bars: A , 0.75 μm; C , 0.5 μm.)

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

    Article Title: Effect of host cell lipid metabolism on alphavirus replication, virion morphogenesis, and infectivity

    doi: 10.1073/pnas.0808720105

    Figure Lengend Snippet: Morphology and physiological features of cholesterol and SM-containing compartments. ( A ) TEM observations illustrating a representative field of the cytoplasm of NHF ( i ) and NPAF ( ii ). Note the abundance of membranous multilamellar inclusions in NPAFs (arrowheads and ii Inset ). m, mitochondria. ( B ) Fluorescence microscopy for analysis of lipid distribution. Staining of NHF ( i ) and NPAF ( ii ) with filipin and phase microscopy ( i′ and ii′ ) show cholesterol-laden organelles in NPAF. Cholesterol and sphingolipid colocalization is demonstrated by filipin ( iii ) and BODIPY-LacCer ( iv ) staining. The merged picture is presented in v (magnification: 100×). ( C ) Fluorescence microscopy ( i–iii ) and TEM ( iv ) showing the endocytic nature of the lipid-containing compartments. Incubation of NPAF with filipin ( i ) to identify cholesterol-containing organelles and with LysoTracker ( ii ) to track the endocytic structures resulted in a partial colocalization of the dyes (see merge in iii ). In iv , NPAF have been incubated with BSA-gold particles (15 nm) for 16 h at 37°C. Arrows highlight BSA-containing structures that morphologically resemble cholesterol and SM-containing compartments. (Scale bars: A , 0.75 μm; C , 0.5 μm.)

    Article Snippet: Cells grown in chamber slides (Nalge–Nunc) were stained with the polyene antibiotic filipin (Sigma) to visualize the distribution of β-hydroxysterols ( , ), with LysoTracker (Molecular Probes) to identify acidic cellular compartments, and with BODIPY-labeled lactosyl ceramide (B-LacCer; Molecular Probes) to visualize the distribution of sphingolipids ( ).

    Techniques: Transmission Electron Microscopy, Fluorescence, Microscopy, Staining, Incubation

    DENV infection stimulates the intracellular cholesterol accumulation at replicative complexes through the activation of HMGCR. The distribution of intracellular cholesterol levels stained with filipin III complex (blue) , and its co-localization with the viral protein NS4A (green) were evaluated by confocal microscopy in Huh7 cells non-infected, infected with DENV4 and treated with DMSO 0.5% (vehicle), 10 mM metformin or 50 μM lovastatin (HMGCR inhibitor) for 24 h. DENV4 infected cells are marked with (+), and non-infected cells marked with (-) . Nuclei were stained with propidium iodide (red) . Numbers inserted in images indicate the co-localization index between cholesterol and NS4A for that specific infected cell. Graph represents NS4A and cholesterol colocalization values as mean ± S.E of 50 infected cells analyzed from 3 independent experiments. Scale bar 10 μm. Images correspond to one representative experiment.

    Journal: PLoS Pathogens

    Article Title: DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target

    doi: 10.1371/journal.ppat.1006257

    Figure Lengend Snippet: DENV infection stimulates the intracellular cholesterol accumulation at replicative complexes through the activation of HMGCR. The distribution of intracellular cholesterol levels stained with filipin III complex (blue) , and its co-localization with the viral protein NS4A (green) were evaluated by confocal microscopy in Huh7 cells non-infected, infected with DENV4 and treated with DMSO 0.5% (vehicle), 10 mM metformin or 50 μM lovastatin (HMGCR inhibitor) for 24 h. DENV4 infected cells are marked with (+), and non-infected cells marked with (-) . Nuclei were stained with propidium iodide (red) . Numbers inserted in images indicate the co-localization index between cholesterol and NS4A for that specific infected cell. Graph represents NS4A and cholesterol colocalization values as mean ± S.E of 50 infected cells analyzed from 3 independent experiments. Scale bar 10 μm. Images correspond to one representative experiment.

    Article Snippet: Intracellular cholesterol was stained with the fluorescent dye filipin III complex (Sigma) and nuclei were counterstained with Hoechst 33342 or propidium iodide (Life Technologies).

    Techniques: Infection, Activation Assay, Staining, Confocal Microscopy

    Reduction in cholesterol accumulation induced by ARF6-GTP expression is coupled to endogenous ARF6-GTP levels in NPC mutant fibroblasts. A , NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with pIRES-GFP bearing ARF6(Q67L). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored blue in merged image) and counterstained with rhodamine phalloidin (red in merged image). Transfected cells are marked with asterisks in filipin images. Note decreased filipin staining in GFP-positive cells. B , NPC mutant fibroblasts (GM03123, GM17923 and GM18436) were treated as in A . Transfected cells with filipin intensity that was lower than neighboring non-transfected cells (as depicted in A ) were scored (see Methods ). 80–100 transfected cells of each cell line were counted in each of three independent experiments. The average percentage of transfected cells displaying reduced filipin intensity is graphed. Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM17923, p = 0.0117 and GM03123 vs. GM18436, p = 0.0114. C , GM03123 cells were transfected with pIRES-GFP bearing ARF6(Q67L) (top row) or ARF6(T27N) (bottom row). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored green in merged image) and immunolabeled for Lamp1 (red in merged image). Bars, 20 µm. Arrow points to region where filipin staining no longer overlaps with Lamp1. D , Lysates were prepared from normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, GM18436) and subjected to the GST-MT-2 pull-down assay and probed for ARF6. Top row is ARF6 precipitated with GST-MT-2 beads and bottom row is ARF6 from the total cell lysate. E , Immunoblots from three independent experiments were subjected to densitometric analysis and the ARF6-GTP/total ARF6 ratios were calculated and normalized to control levels (GM05659). Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM05659, p = 0.011 and GM18436 vs. GM05659, p = 0.022.

    Journal: PLoS ONE

    Article Title: ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease

    doi: 10.1371/journal.pone.0005193

    Figure Lengend Snippet: Reduction in cholesterol accumulation induced by ARF6-GTP expression is coupled to endogenous ARF6-GTP levels in NPC mutant fibroblasts. A , NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with pIRES-GFP bearing ARF6(Q67L). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored blue in merged image) and counterstained with rhodamine phalloidin (red in merged image). Transfected cells are marked with asterisks in filipin images. Note decreased filipin staining in GFP-positive cells. B , NPC mutant fibroblasts (GM03123, GM17923 and GM18436) were treated as in A . Transfected cells with filipin intensity that was lower than neighboring non-transfected cells (as depicted in A ) were scored (see Methods ). 80–100 transfected cells of each cell line were counted in each of three independent experiments. The average percentage of transfected cells displaying reduced filipin intensity is graphed. Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM17923, p = 0.0117 and GM03123 vs. GM18436, p = 0.0114. C , GM03123 cells were transfected with pIRES-GFP bearing ARF6(Q67L) (top row) or ARF6(T27N) (bottom row). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored green in merged image) and immunolabeled for Lamp1 (red in merged image). Bars, 20 µm. Arrow points to region where filipin staining no longer overlaps with Lamp1. D , Lysates were prepared from normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, GM18436) and subjected to the GST-MT-2 pull-down assay and probed for ARF6. Top row is ARF6 precipitated with GST-MT-2 beads and bottom row is ARF6 from the total cell lysate. E , Immunoblots from three independent experiments were subjected to densitometric analysis and the ARF6-GTP/total ARF6 ratios were calculated and normalized to control levels (GM05659). Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM05659, p = 0.011 and GM18436 vs. GM05659, p = 0.022.

    Article Snippet: Reagents Filipin III was obtained from Sigma or Cayman Chemical.

    Techniques: Expressing, Mutagenesis, Transfection, Staining, Immunolabeling, Pull Down Assay, Western Blot

    Constitutively active ARF6 increases cholesterol removal in NPC-like cells. A , HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B , HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C , Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods ). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).

    Journal: PLoS ONE

    Article Title: ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease

    doi: 10.1371/journal.pone.0005193

    Figure Lengend Snippet: Constitutively active ARF6 increases cholesterol removal in NPC-like cells. A , HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B , HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C , Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods ). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).

    Article Snippet: Reagents Filipin III was obtained from Sigma or Cayman Chemical.

    Techniques: Staining, Transfection, Mutagenesis, Expressing, Quantitation Assay, Two Tailed Test

    AhR modulates basal cholesterol levels in fibroblasts. (A) T-FGM AhR+/+ and AhR−/− cultures were stained with the cholesterol dye M1 ganglioside (GM1) and membrane microdomains analyzed by fluorescence confocal microscopy. (B,C) T-FGM cells (B) and primary dermal fibroblasts (C) of both genotypes were grown on glass coverslips, fixed and stained with the cholesterol-binding antibiotic filipin III in order to detect endogenous free cholesterol. Stained cells were analyzed by flow cytometry and the fluorescence intensity profiles represented. (D,E) T-FGM AhR+/+ and AhR−/− cells were incubated for 16 h with 10 mM MβCD, 100 mM cholesterol plus 2.5 mM MβCD (cholesterol) or solvent and analyzed for cholesterol content by flow cytometry as indicated above. Fluorescence profiles were compared graphically. (F) T-FGM cells of both genotypes were grown to confluence and treated with MβCD or cholesterol as indicated above. Wound healing was used to induce directional migration. Cav-1 distribution was analyzed by fluorescence confocal microscopy in a Fluoview F1000 equipment. DAPI staining was used to label cell nuclei. Arrowheads mark Cav-1 location. The experiments were done in duplicate in two cultures of each genotype.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol

    doi: 10.1186/s12964-014-0057-7

    Figure Lengend Snippet: AhR modulates basal cholesterol levels in fibroblasts. (A) T-FGM AhR+/+ and AhR−/− cultures were stained with the cholesterol dye M1 ganglioside (GM1) and membrane microdomains analyzed by fluorescence confocal microscopy. (B,C) T-FGM cells (B) and primary dermal fibroblasts (C) of both genotypes were grown on glass coverslips, fixed and stained with the cholesterol-binding antibiotic filipin III in order to detect endogenous free cholesterol. Stained cells were analyzed by flow cytometry and the fluorescence intensity profiles represented. (D,E) T-FGM AhR+/+ and AhR−/− cells were incubated for 16 h with 10 mM MβCD, 100 mM cholesterol plus 2.5 mM MβCD (cholesterol) or solvent and analyzed for cholesterol content by flow cytometry as indicated above. Fluorescence profiles were compared graphically. (F) T-FGM cells of both genotypes were grown to confluence and treated with MβCD or cholesterol as indicated above. Wound healing was used to induce directional migration. Cav-1 distribution was analyzed by fluorescence confocal microscopy in a Fluoview F1000 equipment. DAPI staining was used to label cell nuclei. Arrowheads mark Cav-1 location. The experiments were done in duplicate in two cultures of each genotype.

    Article Snippet: Free cholesterol measurements T-FGM and primary dermal fibroblasts were fixed with 2% paraformaldehyde for 10 min, washed with PBS and incubated for 30 min at room temperature with 5 μg/ml of the cholesterol-binding filipin III (Sigma).

    Techniques: Staining, Fluorescence, Confocal Microscopy, Binding Assay, Flow Cytometry, Cytometry, Incubation, Migration

    The fluorescent images of PAMAM–PEG–SRL/ EMA-labeled DNA cellular internalization. Internalization mechanism of nanoparticles by C6 glioma cells. Concentration of PAMAM of all samples was adjusted to 1μM (250 μg/m). PAMAM–PEG–SRL/EMA-labeled DNA without any inhibitor (as control) (A, B). Cells were incubated with different endocytosis inhibitors including: lactoferrin (E, F), phenylarsine oxide (G, H), filipin complex (I, J), colchicine (K, L), and at 4 o C (C, D); for 30 min. Red: EMA-labeled DNA. Bar: 50μm

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    Article Title: SRL-Coated PAMAM Dendrimer Nano-Carrier for Targeted Gene Delivery to the Glioma Cells and Competitive Inhibition by Lactoferrin

    doi:

    Figure Lengend Snippet: The fluorescent images of PAMAM–PEG–SRL/ EMA-labeled DNA cellular internalization. Internalization mechanism of nanoparticles by C6 glioma cells. Concentration of PAMAM of all samples was adjusted to 1μM (250 μg/m). PAMAM–PEG–SRL/EMA-labeled DNA without any inhibitor (as control) (A, B). Cells were incubated with different endocytosis inhibitors including: lactoferrin (E, F), phenylarsine oxide (G, H), filipin complex (I, J), colchicine (K, L), and at 4 o C (C, D); for 30 min. Red: EMA-labeled DNA. Bar: 50μm

    Article Snippet: Lactoferrin, filipin complex (from Streptomyces filipinesis), L-polylysin (30000-70000MW) and phenylarsine oxide were bought from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Labeling, Concentration Assay, Incubation

    Filipin treatment affects activation of the HOG pathway. (A) Filipin treatment impedes activation of the Sho1 branch. Wild-type (TM141, WT), ssk2 , 22 Δ (TM252), and sho1 Δ (TM287) cells were treated or not treated with 5 μg/ml filipin

    Journal: Molecular and Cellular Biology

    Article Title: Sphingolipids Regulate the Yeast High-Osmolarity Glycerol Response Pathway

    doi: 10.1128/MCB.06111-11

    Figure Lengend Snippet: Filipin treatment affects activation of the HOG pathway. (A) Filipin treatment impedes activation of the Sho1 branch. Wild-type (TM141, WT), ssk2 , 22 Δ (TM252), and sho1 Δ (TM287) cells were treated or not treated with 5 μg/ml filipin

    Article Snippet: Five micrograms/milliliter filipin complex (Sigma) was added to the logarithmically growing YPD culture of TM141, TM252 ( ssk2 Δ ssk22 Δ), or TM287 ( sho1 Δ) cells.

    Techniques: Activation Assay

    Distribution of cholesterol in membranes of normal human dermal fibroblasts NHDF (left) and melanoma cells A375 (right). Cholesterol was stained with filipin (green) and nucleus with PI (red). Overlay at the bottom. Pictures are representative results of more than three independent experiments.

    Journal: PLoS ONE

    Article Title: Interaction of two antitumor peptides with membrane lipids – Influence of phosphatidylserine and cholesterol on specificity for melanoma cells

    doi: 10.1371/journal.pone.0211187

    Figure Lengend Snippet: Distribution of cholesterol in membranes of normal human dermal fibroblasts NHDF (left) and melanoma cells A375 (right). Cholesterol was stained with filipin (green) and nucleus with PI (red). Overlay at the bottom. Pictures are representative results of more than three independent experiments.

    Article Snippet: Cholesterol staining Filipin complex (Sigma-Aldrich Co. LLC) (excitation wavelength 352 nm; emission wavelength 454 nm) was used for cholesterol staining of cell membranes of NHDF and A375.

    Techniques: Staining

    Influence of cholesterol depletion of plasma membrane (PM) on killing efficiency of peptides. A375 cells were treated with 0 mM, 5 mM and 10 mM of cholesterol depletion agent MβCD before peptide incubation. Top: Cholesterol of plasma membrane PM (left) was already efficiently depleted in the presence of 5 mM MβCD (right) as seen by microscopic inspection upon staining with filipin (green). Only intracellular cholesterol remained, presumably located to the Golgi apparatus (GA). Bottom: Upon peptide incubation (10 μM and 20 μM DIM-LF11-322, left; 10 and 20 μM R-DIM-P-LF11-318, right) the PI-uptake of cancer cells, which is proportional to the percentage of killed cells, was measured over four hours. Black represents cell death of A375 in presence of peptides without MβCD treatment, where plasma membrane cholesterol is present. Light gray and gray dashed lines represent cells treated with 5 mM and 10 mM MβCD, respectively. The result indicates a reduction of R-DIM-P-LF11-322 efficacy in presence of PM cholesterol, whereas toxicity of DIM-LF11-318 seems independent on PM cholesterol. Both peptides do not enter cells by clathrin or caveolae mediated endocytosis. Pictures are representative results of three independent experiments. PI-uptake is shown as a mean ± SEM of three independent experiments.

    Journal: PLoS ONE

    Article Title: Interaction of two antitumor peptides with membrane lipids – Influence of phosphatidylserine and cholesterol on specificity for melanoma cells

    doi: 10.1371/journal.pone.0211187

    Figure Lengend Snippet: Influence of cholesterol depletion of plasma membrane (PM) on killing efficiency of peptides. A375 cells were treated with 0 mM, 5 mM and 10 mM of cholesterol depletion agent MβCD before peptide incubation. Top: Cholesterol of plasma membrane PM (left) was already efficiently depleted in the presence of 5 mM MβCD (right) as seen by microscopic inspection upon staining with filipin (green). Only intracellular cholesterol remained, presumably located to the Golgi apparatus (GA). Bottom: Upon peptide incubation (10 μM and 20 μM DIM-LF11-322, left; 10 and 20 μM R-DIM-P-LF11-318, right) the PI-uptake of cancer cells, which is proportional to the percentage of killed cells, was measured over four hours. Black represents cell death of A375 in presence of peptides without MβCD treatment, where plasma membrane cholesterol is present. Light gray and gray dashed lines represent cells treated with 5 mM and 10 mM MβCD, respectively. The result indicates a reduction of R-DIM-P-LF11-322 efficacy in presence of PM cholesterol, whereas toxicity of DIM-LF11-318 seems independent on PM cholesterol. Both peptides do not enter cells by clathrin or caveolae mediated endocytosis. Pictures are representative results of three independent experiments. PI-uptake is shown as a mean ± SEM of three independent experiments.

    Article Snippet: Cholesterol staining Filipin complex (Sigma-Aldrich Co. LLC) (excitation wavelength 352 nm; emission wavelength 454 nm) was used for cholesterol staining of cell membranes of NHDF and A375.

    Techniques: Incubation, Staining

    Changes in distribution of intracellular and plasma membrane cholesterol in cells of melanoma A375 upon peptide addition. A375 with stained cholesterol (filipin, green) were treated with 10 μM R-DIM-P-LF11-322 or DIM-LF11-318 for indicated time periods. Initial localization of cholesterol in PM and GA in untreated melanoma cells changes similarly, however with increased velocity upon treatment with DIM-LF11-318. Both peptides induce consumption of intracellular cholesterol and appearance of cholesterol rich domains in the plasma membrane after 30 min (DIM-LF11-318) or 60–120 min (R-DIM-P-LF11-322). Bottom OL BF: Overlay of bright field (OL BF) and fluorescence (green/filipin, staining of cholesterol; red/PI, staining of DNA of membrane damaged cells) upon 120 min of respective peptide incubation. DIM-LF11-318 causes release of small beads (white arrows), partially composed of cholesterol (green staining). Arrows indicate the plasma membrane (PM), Golgi apparatus (GA) as well as domains (DOM) and beads. Pictures are representative for a series of three experiments.

    Journal: PLoS ONE

    Article Title: Interaction of two antitumor peptides with membrane lipids – Influence of phosphatidylserine and cholesterol on specificity for melanoma cells

    doi: 10.1371/journal.pone.0211187

    Figure Lengend Snippet: Changes in distribution of intracellular and plasma membrane cholesterol in cells of melanoma A375 upon peptide addition. A375 with stained cholesterol (filipin, green) were treated with 10 μM R-DIM-P-LF11-322 or DIM-LF11-318 for indicated time periods. Initial localization of cholesterol in PM and GA in untreated melanoma cells changes similarly, however with increased velocity upon treatment with DIM-LF11-318. Both peptides induce consumption of intracellular cholesterol and appearance of cholesterol rich domains in the plasma membrane after 30 min (DIM-LF11-318) or 60–120 min (R-DIM-P-LF11-322). Bottom OL BF: Overlay of bright field (OL BF) and fluorescence (green/filipin, staining of cholesterol; red/PI, staining of DNA of membrane damaged cells) upon 120 min of respective peptide incubation. DIM-LF11-318 causes release of small beads (white arrows), partially composed of cholesterol (green staining). Arrows indicate the plasma membrane (PM), Golgi apparatus (GA) as well as domains (DOM) and beads. Pictures are representative for a series of three experiments.

    Article Snippet: Cholesterol staining Filipin complex (Sigma-Aldrich Co. LLC) (excitation wavelength 352 nm; emission wavelength 454 nm) was used for cholesterol staining of cell membranes of NHDF and A375.

    Techniques: Staining, Fluorescence, Incubation

    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

    ( See previous page ). MYO1C-depleted cells contain more total cellular cholesterol, trapped in intracellular storage compartments. ( A ) Confocal z-projection microscopy images of cellular cholesterol, imaged with filipin, from mock and MYO1C siRNA-depleted HeLa cells. Scale bar = 20 μm ( B ) Total cholesterol levels in mock and MYO1C-knockdown cells and DMSO- and PClP-treated cells were quantified by high-throughput microscopy. Automated imaging and analysis software was used to calculate the total filipin fluorescence per cell. Loss of MYO1C caused a significant increase in cholesterol as compared to control cells. In total > 149 ,000 cells from 3 independent experiments, each performed in triplicate, were analyzed. Graphs represent the means ± s.e.m. ( C ) Model of the autophagy and endocytic pathway highlighting defects (shown in blue) observed in MYO1C-depleted cells. Loss of functional MYO1C causes a defect in lipid raft recycling from the perinuclear recycling compartment back to the cell surface. This leads to intracellular accumulation of cholesterol-enriched membranes. 6 In the classical endocytic pathway, incoming cargo first moves through early endosomes, which then acquire an increasing number of intralumenal vesicles and mature into late endosomes (LE)/multivesicular bodies (MVB). The fusion of a LE/MVB with a lysosome generates a transient hybrid organelle, the endolysosome, in which content degradation can take place. In MYO1C-depleted cells, we observe the accumulation of enlarged LAMP1- and CTSD-positive endolysosomes. In the autophagy pathway, cytosolic material is sequestered by expansion and closure of a phagophore, forming double-membrane vesicles called autophagosomes. These autophagosomes mature by fusion with early and late endosomes to form an intermediate organelle called the amphisome, which fuses with lysosomes to enable content degradation. Ablating MYO1C activity leads to an accumulation in the number of autophagic structures suggesting a block in fusion of autophagic organelles with lysosomes.

    Journal: Autophagy

    Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion

    doi: 10.4161/15548627.2014.984272

    Figure Lengend Snippet: ( See previous page ). MYO1C-depleted cells contain more total cellular cholesterol, trapped in intracellular storage compartments. ( A ) Confocal z-projection microscopy images of cellular cholesterol, imaged with filipin, from mock and MYO1C siRNA-depleted HeLa cells. Scale bar = 20 μm ( B ) Total cholesterol levels in mock and MYO1C-knockdown cells and DMSO- and PClP-treated cells were quantified by high-throughput microscopy. Automated imaging and analysis software was used to calculate the total filipin fluorescence per cell. Loss of MYO1C caused a significant increase in cholesterol as compared to control cells. In total > 149 ,000 cells from 3 independent experiments, each performed in triplicate, were analyzed. Graphs represent the means ± s.e.m. ( C ) Model of the autophagy and endocytic pathway highlighting defects (shown in blue) observed in MYO1C-depleted cells. Loss of functional MYO1C causes a defect in lipid raft recycling from the perinuclear recycling compartment back to the cell surface. This leads to intracellular accumulation of cholesterol-enriched membranes. 6 In the classical endocytic pathway, incoming cargo first moves through early endosomes, which then acquire an increasing number of intralumenal vesicles and mature into late endosomes (LE)/multivesicular bodies (MVB). The fusion of a LE/MVB with a lysosome generates a transient hybrid organelle, the endolysosome, in which content degradation can take place. In MYO1C-depleted cells, we observe the accumulation of enlarged LAMP1- and CTSD-positive endolysosomes. In the autophagy pathway, cytosolic material is sequestered by expansion and closure of a phagophore, forming double-membrane vesicles called autophagosomes. These autophagosomes mature by fusion with early and late endosomes to form an intermediate organelle called the amphisome, which fuses with lysosomes to enable content degradation. Ablating MYO1C activity leads to an accumulation in the number of autophagic structures suggesting a block in fusion of autophagic organelles with lysosomes.

    Article Snippet: To visualize cholesterol, nonpermeabilized cells were labeled with freshly prepared filipin-complex (Sigma, F4767) in DMSO at a final concentration of 25 μg/ml.

    Techniques: Polyacrylamide Gel Electrophoresis, Microscopy, High Throughput Screening Assay, Imaging, Software, Fluorescence, Functional Assay, Activity Assay, Blocking Assay

    Intracellular cholesterol retention and perturbed membrane electrical properties in PMP22-deficient Schwann cells. A , B , Representative images of WT and PMP22 KO mouse Schwann cells ( A ) and quantification of filipin intensity ( B ) in the Golgi and at the plasma membrane (PM) after costaining with anti-GS15 antibody ( n = 80 cells each genotype). “Rest” indicates the rest of a cell minus the Golgi and PM. C , Representative images of PMP22 (red) and cholesterol (blue) localization after scrambled or PMP22 shRNA-GFP plasmid (green) transfection in rat Schwann cells. Arrows point to transfected cells. D , Images depicting the location of patch pipettes relative to the cells subjected to current-clamp recordings. E – G , Current-clamp recordings of WT (gray bars) and PMP22 KO (black bars) mouse Schwann cells showing the membrane capacitance ( E ), membrane resistance ( F ), and time constant ( G ) for each experimental group ( n = 10 cells per group from three independent replicates). For all experiments, values represent the mean ± SEM. ** p

    Journal: The Journal of Neuroscience

    Article Title: PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux

    doi: 10.1523/JNEUROSCI.2942-18.2019

    Figure Lengend Snippet: Intracellular cholesterol retention and perturbed membrane electrical properties in PMP22-deficient Schwann cells. A , B , Representative images of WT and PMP22 KO mouse Schwann cells ( A ) and quantification of filipin intensity ( B ) in the Golgi and at the plasma membrane (PM) after costaining with anti-GS15 antibody ( n = 80 cells each genotype). “Rest” indicates the rest of a cell minus the Golgi and PM. C , Representative images of PMP22 (red) and cholesterol (blue) localization after scrambled or PMP22 shRNA-GFP plasmid (green) transfection in rat Schwann cells. Arrows point to transfected cells. D , Images depicting the location of patch pipettes relative to the cells subjected to current-clamp recordings. E – G , Current-clamp recordings of WT (gray bars) and PMP22 KO (black bars) mouse Schwann cells showing the membrane capacitance ( E ), membrane resistance ( F ), and time constant ( G ) for each experimental group ( n = 10 cells per group from three independent replicates). For all experiments, values represent the mean ± SEM. ** p

    Article Snippet: For filipin–cholesterol labeling, mouse Schwann cells were fixed with 4% paraformaldehyde for 10 min. After rinsing in PBS, samples were incubated with 50 μg/ml filipin III (F4767; Sigma-Aldrich) in PBS for 1 h in the dark.

    Techniques: shRNA, Plasmid Preparation, Transfection

    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