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  • 95
    Millipore filipin complex
    The cofilin and actin mutants facilitate the enrichment of sterols at the sites of tombusvirus replication in yeast. (A) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA. Fluorescent microscopic images of yeast cells stained with <t>filipin</t> dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free wt yeast cells, while act1-121 ts yeast and act1-132 ts yeast show uneven distribution of ergosterols in the absence of viral components (right images). (B) Similar experiments as in Panel A, except performed at the semi-permissive temperature (27°C) in act1-132 ts and WT yeasts. (C) Similar experiments as in Panel A, except performed at the semi-permissive temperature (32°C) in act1-121 ts and WT yeasts. (D) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the permissive temperature. Fluorescent microscopic images of yeast cells stained with filipin dye. (E) Re-localization of ergosterols to internal punctate structures in yeast replicating TBSV repRNA in cof1-8 ts or wt yeasts at the semi-permissive temperature. See further details in panel A. Each experiment was repeated at least three times.
    Filipin Complex, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical filipin
    CPPs induce <t>filipin-positive</t> puncta in an endocytosis-dependent manner. A and B , HEK293 cells were cultured with BAPTA-AM (10 μ m ), MβCD (2.5 m m ), or dynasore (50 μ m ) for 30 min followed by CPP treatment for 4 h. Cells were then
    Filipin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore filipin
    Electron microscopical visualization of <t>filipin</t> labeled membrane cholesterol. Arrowheads in control (A) and double-deficient fibroblasts (B) indicate the filipin labeling. In control cells labeling was detected in the limiting membranes of small vesicles
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PolyScience filipin
    Surface marker and biochemical characterization of Npc1 microglial. ( A ) Microglia surface marker expression was characterized in Npc1 +/+ (red) and Npc1 −/− (dashed blue) microglia at birth, 3-, 7- and 9-weeks of age. Microglia steady state markers (CD11b, CD45, CX3CR1 and F4/80) and activation markers (CD86 and MHCII) were quantified. ( B ) GM1 and unesterified cholesterol storage as mean fluorescence intensity (MFI) at birth, 3-, 7- and 9-weeks of age in microglia isolated from Npc1 +/+ (red) and Npc1 −/− (dashed blue) mice. GM1 and unesterified cholesterol accumulation was quantified by staining isolated microglial cells with Alexa488-labeled cholera toxin and <t>filipin,</t> respectively, n ≥3.
    Filipin, supplied by PolyScience, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Polysciences inc filipin
    Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by <t>filipin</t> fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P
    Filipin, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology filipin
    HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL <t>filipin</t> (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p
    Filipin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher filipin iii
    Lipid transport out of MVBs is dispensable for CCHFV entry. (A) SW13 cells were pretreated with U18666A (30 µM) for 1 h or left untreated (mock). Then, the cells were incubated with CCHFV in the presence of the drug for 24 h and subsequently fixed, permeabilized, and stained with anti-N antibody (red), anti-CD63 antibody (green), and CellMask blue dye (grey) to define cell boundaries. The samples were imaged by immunofluorescence, and an optical section through the middle of the cell is shown (left and middle panels). Relative infection efficiencies were calculated by dividing the number of infected cells by the total number of cells and are averages of three independent experiments, with error bars representing standard deviations (right panel). (B) Cells treated as described in (A) were fixed 1 h after treatment and then stained with anti-CD63 antibody (green), <t>filipin</t> <t>III</t> (red), and CellMask red dye (grey). The images were generated as described above. (C) SW13 cells were treated with U18666A (30 µM) for 1 h or left untreated (mock), then incubated with VSV-CCHFVG, VSV-EBOVGP, or VSV-LASVGP. Luciferase activity was measured 24 h after pseudotype addition.
    Filipin Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem filipin iii
    Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), <t>filipin</t> <t>III</t> (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m
    Filipin Iii, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Merck & Co filipin iii
    Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), <t>filipin</t> <t>III</t> (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m
    Filipin Iii, supplied by Merck & Co, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    AG Scientific filipin complex
    Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), <t>filipin</t> <t>III</t> (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m
    Filipin Complex, supplied by AG Scientific, used in various techniques. Bioz Stars score: 84/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore prepared filipin
    GP73 regulates the transcriptional activity of SREBPs and lipogenesis. ( a ) Immunoblotting analysis of SREBPs activation in HepG2 cells transfected with Flag-GP73 at the indicated doses. α-Tubulin was used as equal loading control. ( b , c ) SREBP-1 promoter activity in HepG2 ( b ) or HL7702 ( c ) cells transfected with Flag-vector or Flag-GP73 under conditions of sterol depletion or repletion. The luciferase activity was measured 36 hrs post transfection. The value was normalized with the corresponding transfection efficiency. ( d , f , h ) QRT-PCR analysis of HMGR ( d ), FASN2 ( f ), and ACC1 ( h ) mRNA abundance in HepG2 cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( e , g , i ) QRT-PCR analysis of HMGR ( e ), FASN2 ( g ), and ACC1 ( i ) mRNA abundance in 293T cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( j ) Fluorescence microscopy of <t>Filipin</t> staining in HepG2 cells transfected with Flag-GP73. Cells were collected at indicated hrs post transfection. ( k , l ) Amplex Red cholesterol assay of cellular cholesterol concentrations in HepG2 ( k ) or HL7702 ( l ) cells transfected with Flag-vector or Flag-GP73. Cells were collected at indicated hrs post transfection. Values were normalized to total cell proteins from control cells transfected with Flag-vector. Cell-based studies were performed at least <t>three</t> independent times with comparable results. Data represent mean ± SEM. Student’s t test was used for statistical analysis: **p
    Prepared Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Omega Optical 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
    Filipin Iii, supplied by Omega Optical, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biomol GmbH filipin iii
    IL-36 signaling modulates cholesterol metabolism in Mtb -infected macrophage. ( A ) Changes in total cholesterol, free cholesterol and cholesteryl ester in cell lysates from scramble and IL36R KD THP-1 macrophages upon Mtb infection. ( B ) Immunofluorescence staining with <t>Filipin</t> <t>III</t> in non-infected (N/I), 24 h and 48 h Mtb -infected scramble versus IL36R KD THP-1 macrophages (40× objective magnification). ( C ) mRNA expression of CD36 , LDLR , SRA in Mtb -infected scramble versus IL36R KD cells. ( D ) Cholesterol efflux upon Mtb infection or LXR stimulation in scramble versus IL36R KD THP-1 macrophages with or without LXR inhibitors. ( E ) SREBP2 protein expression in Mtb -infected scramble and IL-36 deficient cells. ( F ) mRNA expression of several cholesterol synthesis genes in Mtb -infected macrophages at 30 h with or without 27HC, 25HC and betulin stimulation. ( A , C , D , F ) Data pooled from three independent experiments are shown. Data are shown as mean ± SD. ( B and E ) Data from one representative experiment of at least two independent experiments are shown. P values: ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Filipin Iii, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Millipore filipin dye
    Fluorescence imaging confirms differences in MCA smooth muscle <t>CLR</t> levels among experimental groups. A. Original snapshots showing vascular smooth muscle CLR levels via fluorescence staining of arteries with CLR-sensitive dye <t>filipin.</t> Arteries were collected from rats on high CLR diet + placebo, and high CLR diet + atorvastatin. Several arteries from rats on high CLR diet + atorvastatin were subjected to in vitro CLR enrichment. Artery staining from all experimental groups was performed in parallel to minimize the experimental error and justify the direct comparison of the fluorescence signal intensity. Snapshots on the right show smooth muscle layer in visible light. These images were superposed with fluorescence ones to define the individual myocyte plasma membranes for filipin intensity quantification. In the right top panel, arrows point at the example of individual myocyte’s silhouette (light grey) within vasculature layer. B. Averaged data showing filipin fluorescence signal from high CLR diet + placebo (n = 7), high CLR diet + atorvastatin (n = 10), and high CLR diet + atorvastatin subjected to CLR enrichment in vitro (n = 8). (“n” refers to number of artery segments). Here and in C, *statistically significant difference; P
    Filipin Dye, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    BioVision filipin iii signals
    ASM inhibitors induce melanoma-specific cell death by inhibiting intracellular cholesterol transport. ( A ) fluorescence microscopy of intracellular cholesterol localisation following knockdown of SMPD1 (ASM1) as detected by <t>Filipin-III</t> staining (arrows show accumulated cholesterol); ( B ) the viability of UACC 903 and 1205 Lu melanoma cells following knockdown of SMPD1; ( C ) IC 50 values of various melanoma cell lines and FF2441 fibroblasts treated with ASM inhibitors. Dose–response curves were drawn in OriginPro (OriginLab) using Levenberg Marquardt algorithm; ( D ) viability of UACC 903 and FF2441 cells following 24 h of treatment with increasing concentrations of ASM inhibitors; ( E ) viability of melanoma cells treated with various ASM inhibitors in the absence or presence of v-ATPase inhibitor, Bafilomycin-A1; ( F ) intracellular cholesterol localisation following leelamine, U18666A or ASM inhibitor treatments as detected by Filipin-III staining. (lower) Co-localisation of RFP-tagged lysosomal LAMP1 protein with cholesterol; ( G ) LDL treatment protects UACC 903 cells from ASM inhibitor-mediated cell death; ( H ) Venn diagram showing number of significantly altered genes identified by RNA-sequencing of UACC 903 cells treated with ASM inhibitors (left); and enrichment analysis of significantly altered 177 genes; ( I ) expression level of cholesterol synthesis genes following ASM treatment as detected by RNA-sequencing; ( J ) cholesterol synthesis pathway. White highlighted genes were identified as significantly deregulated by RNA-sequencing; ( K ) distribution of log-2-fold change in expression levels of cholesterol synthesis genes identified by microarray analysis following treatment of MCF7 cells with ASM inhibitors.
    Filipin Iii Signals, supplied by BioVision, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Polysciences inc filipin solution
    Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) <t>Filipin</t> staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p
    Filipin Solution, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Abcam filipin
    Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. <t>Filipin</t> staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p
    Filipin, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    FUJIFILM 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>
    Filipin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ApexBio 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>
    Filipin, supplied by ApexBio, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Zeiss filipin
    IGF2 induces lipid droplet formation but has no influence on lipid droplet size. (A) Representative LD540 fluorescence staining for lipid droplets (LD, yellow droplets) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were stained with DAPI (blue) (scale bar: 20 μm) (upper panel). Five randomly selected pictures per sample were collected and analyzed with ImageJ. Counted LDs were normalized to the nuclei count (lower panel, left ). Total LD area was normalized to the nuclei count (lower panel, middle ). Total LD size was estimated as the mean area of all counted LDs (lower panel, right ) (co, n = 10; Igf2, n = 11, independent two-sample t -test). (B) Representative <t>filipin</t> fluorescence staining for free cholesterol (black droplets) in Igf2 and co animals (co, n = 10; Igf2, n = 11, arrows designate examples for free cholesterol). (C) Levels of Hmgcr mRNA, real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA and co. All results are presented as mean ± SEM.
    Filipin, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 90/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Genmed filipin staining kit
    Quercetin and lipid accumulation in RAW264.7 cells induced by ox-LDL. (A) RAW264.7 cells were treated with quercetin and ox-LDL and the lipid accumulation was measured by oil red staining. (B) RAW264.7 cells were treated with quercetin and ox-LDL and the intracellular free cholesterol was measured using <t>Filipin</t> staining. (C) RAW264.7 cells were treated with quercetin and ox-LDL and the total cellular cholesterol was measured. Data are presented as the mean ± standard deviation. *P
    Filipin Staining Kit, supplied by Genmed, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Millipore sterol binding compound filipin
    Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with <t>filipin.</t> The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type
    Sterol Binding Compound Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genmed filipin fluorescence staining kit
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Filipin Fluorescence Staining Kit, supplied by Genmed, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lipid raft inhibitor filipin iii
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Lipid Raft Inhibitor Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Cayman Chemical filipin stain
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Filipin Stain, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Vector Laboratories filipin dapi
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Filipin Dapi, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore filipin pbs
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Filipin Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Cayman Chemical filipin staining
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Filipin Staining, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 87/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Cayman Chemical filipin based detection kit
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
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    Image Search Results


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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1005440

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

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

    Techniques: Staining

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

    Journal: Fungal Genetics and Biology

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

    doi: 10.1016/j.fgb.2011.11.006

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

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

    Techniques: Staining

    CPPs induce filipin-positive puncta in an endocytosis-dependent manner. A and B , HEK293 cells were cultured with BAPTA-AM (10 μ m ), MβCD (2.5 m m ), or dynasore (50 μ m ) for 30 min followed by CPP treatment for 4 h. Cells were then

    Journal: The Journal of Biological Chemistry

    Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *

    doi: 10.1074/jbc.M113.531855

    Figure Lengend Snippet: CPPs induce filipin-positive puncta in an endocytosis-dependent manner. A and B , HEK293 cells were cultured with BAPTA-AM (10 μ m ), MβCD (2.5 m m ), or dynasore (50 μ m ) for 30 min followed by CPP treatment for 4 h. Cells were then

    Article Snippet: Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software.

    Techniques: Cell Culture, Conditioned Place Preference

    Additional NMJ images and quantifications of bouton number and bouton size. ( A ) Quantification of bouton number of NMJ4 from wild-type larvae fed with 0 mM (CT), 10 mM, 20 mM MβCD and 0 μg/ml (DMSO vehicle only), 25 μg/ml, 50 μg/ml filipin III. ( B and C ) Quantifications of muscle four surface area ( B ), relative bouton number and size normalized to muscle surface area ( C ) upon indicated treatments. n ≥ 8 larvae; ns, no significance, *p

    Journal: eLife

    Article Title: The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt

    doi: 10.7554/eLife.38183

    Figure Lengend Snippet: Additional NMJ images and quantifications of bouton number and bouton size. ( A ) Quantification of bouton number of NMJ4 from wild-type larvae fed with 0 mM (CT), 10 mM, 20 mM MβCD and 0 μg/ml (DMSO vehicle only), 25 μg/ml, 50 μg/ml filipin III. ( B and C ) Quantifications of muscle four surface area ( B ), relative bouton number and size normalized to muscle surface area ( C ) upon indicated treatments. n ≥ 8 larvae; ns, no significance, *p

    Article Snippet: D,L-threo-PDMP (Matreya) and filipin III (Cayman) were dissolved in DMSO and then individually added to standard media at specific concentrations.

    Techniques:

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

    Journal: Molecular Biology of the Cell

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

    doi: 10.1091/mbc.E04-02-0103

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

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

    Techniques: Labeling

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

    Journal: Molecular Biology of the Cell

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

    doi: 10.1091/mbc.E04-02-0103

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

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

    Techniques: Expressing, Transfection, Staining

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

    Journal: Molecular Biology of the Cell

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

    doi: 10.1091/mbc.E04-02-0103

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

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

    Techniques: Staining

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

    Journal: Molecular Biology of the Cell

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

    doi: 10.1091/mbc.E04-02-0103

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

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

    Techniques: Staining

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

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

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

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

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

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

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

    Techniques: Incubation, Staining, Labeling, Fluorescence

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

    Techniques: Labeling, Staining, Marker, Two Tailed Test

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

    Techniques: Staining, Labeling, Confocal Microscopy

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

    Journal: The EMBO Journal

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

    doi: 10.15252/embj.201695917

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

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

    Techniques: Labeling, Staining, Fluorescence

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0029537

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0029537

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

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

    Techniques: Fluorescence, Staining, Cell Culture, Incubation

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

    Journal: Journal of Neurophysiology

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

    doi: 10.1152/jn.00779.2011

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

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

    Techniques: Staining

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

    Journal: Infection and Immunity

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

    doi: 10.1128/IAI.01980-14

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

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

    Techniques: Incubation, Labeling

    Surface marker and biochemical characterization of Npc1 microglial. ( A ) Microglia surface marker expression was characterized in Npc1 +/+ (red) and Npc1 −/− (dashed blue) microglia at birth, 3-, 7- and 9-weeks of age. Microglia steady state markers (CD11b, CD45, CX3CR1 and F4/80) and activation markers (CD86 and MHCII) were quantified. ( B ) GM1 and unesterified cholesterol storage as mean fluorescence intensity (MFI) at birth, 3-, 7- and 9-weeks of age in microglia isolated from Npc1 +/+ (red) and Npc1 −/− (dashed blue) mice. GM1 and unesterified cholesterol accumulation was quantified by staining isolated microglial cells with Alexa488-labeled cholera toxin and filipin, respectively, n ≥3.

    Journal: Human Molecular Genetics

    Article Title: Microglia activation in Niemann–Pick disease, type C1 is amendable to therapeutic intervention

    doi: 10.1093/hmg/ddy112

    Figure Lengend Snippet: Surface marker and biochemical characterization of Npc1 microglial. ( A ) Microglia surface marker expression was characterized in Npc1 +/+ (red) and Npc1 −/− (dashed blue) microglia at birth, 3-, 7- and 9-weeks of age. Microglia steady state markers (CD11b, CD45, CX3CR1 and F4/80) and activation markers (CD86 and MHCII) were quantified. ( B ) GM1 and unesterified cholesterol storage as mean fluorescence intensity (MFI) at birth, 3-, 7- and 9-weeks of age in microglia isolated from Npc1 +/+ (red) and Npc1 −/− (dashed blue) mice. GM1 and unesterified cholesterol accumulation was quantified by staining isolated microglial cells with Alexa488-labeled cholera toxin and filipin, respectively, n ≥3.

    Article Snippet: Filipin 0.1% (m/v) (Polyscience, Warrington, USA) and FITC-conjugated cholera toxin (1/300) were incubated for 2 h at 4°C before being washed twice with BD wash buffer.

    Techniques: Marker, Expressing, Activation Assay, Fluorescence, Isolation, Mouse Assay, Staining, Labeling

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0074169

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

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

    Techniques: Activation Assay, Fluorescence

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0074169

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

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

    Techniques: Western Blot, Staining, Fluorescence

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0074169

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

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

    Techniques: Expressing, Transfection, Staining

    HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

    doi: 10.1016/j.omtm.2018.12.005

    Figure Lengend Snippet: HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization (A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method , followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p

    Article Snippet: Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada).

    Techniques: Transfection, Neutralization, Incubation, Microscopy, Software, Lactate Dehydrogenase Assay

    EMC loss does not activate a cholesterol homeostatic response. (A) Isolation of cytoplasmic (C) and nuclear (N) fractions from WT and ΔEMC6 (Δ6) cells by subcellular fractionation. Cells were treated with MBCD (1 mM), 25-hydroxycholesterol (25HC, 25 µM) or ethanol (control, Veh.) for 4 h. Western blots probing for the full-length (FL) and cleaved N-terminal fragment (N) of SREBP-2 are shown, along with Sec61β and histone H3 as controls for cytoplasmic and nuclear fractions, respectively. The asterisk (*) denotes a nonspecific band. (B) mRNA levels of HMGCR, SQS and SM as determined by quantitative real-time RT-PCR (qRT-PCR). Transcript levels present in untreated ΔEMC5 (Δ5) and Δ6 U2OS Flp-In™ T-Rex™ cells relative to WT are shown as means±s.d. ( n =6). (C) Western blots of whole-cell lysates derived from WT, Δ5 and Δ6 cells grown in normal medium were probed with the indicated antibodies, where Hsp70 served as a loading control. (D) Filipin III staining of WT and Δ6 cells maintained in DMEM plus 10% FCS to monitor intracellular cholesterol distribution. (E) mRNA levels of HMGCR, SM and SQS from WT and Δ6 cells treated with 1 mM MBCD for 0, 2 and 4 h, as determined by qRT-PCR relative to WT. Means±s.d. are shown ( n =3).

    Journal: Journal of Cell Science

    Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis

    doi: 10.1242/jcs.223453

    Figure Lengend Snippet: EMC loss does not activate a cholesterol homeostatic response. (A) Isolation of cytoplasmic (C) and nuclear (N) fractions from WT and ΔEMC6 (Δ6) cells by subcellular fractionation. Cells were treated with MBCD (1 mM), 25-hydroxycholesterol (25HC, 25 µM) or ethanol (control, Veh.) for 4 h. Western blots probing for the full-length (FL) and cleaved N-terminal fragment (N) of SREBP-2 are shown, along with Sec61β and histone H3 as controls for cytoplasmic and nuclear fractions, respectively. The asterisk (*) denotes a nonspecific band. (B) mRNA levels of HMGCR, SQS and SM as determined by quantitative real-time RT-PCR (qRT-PCR). Transcript levels present in untreated ΔEMC5 (Δ5) and Δ6 U2OS Flp-In™ T-Rex™ cells relative to WT are shown as means±s.d. ( n =6). (C) Western blots of whole-cell lysates derived from WT, Δ5 and Δ6 cells grown in normal medium were probed with the indicated antibodies, where Hsp70 served as a loading control. (D) Filipin III staining of WT and Δ6 cells maintained in DMEM plus 10% FCS to monitor intracellular cholesterol distribution. (E) mRNA levels of HMGCR, SM and SQS from WT and Δ6 cells treated with 1 mM MBCD for 0, 2 and 4 h, as determined by qRT-PCR relative to WT. Means±s.d. are shown ( n =3).

    Article Snippet: Chemicals and compounds The following compounds were used: 25-hydroxycholesterol (25HC, Sigma-Aldrich, St. Louis, MO), DAPI (Sigma-Aldrich), aminooxybiotin (Biotium, Fremont, CA), aniline hydrochloride (Sigma-Aldrich), avasimibe (Sigma-Aldrich), cholesterol (Sigma-Aldrich), doxycycline (Sigma-Aldrich), filipin III (Santa Cruz Biotechnology, Dallas, TX), gel filtration markers kit (MWGF1000, Sigma-Aldrich), GGTI 298 trifluoroacetate salt hydrate (Sigma-Aldrich), Hygromycin B Gold (InvivoGen, San Diego, CA), IAA (iodoacetamide, Sigma-Aldrich), lipoprotein-depleted fetal bovine serum (LPDS, Kalen Biomedical, Germantown, MD), LMNG (lauryl maltose neopentyl glycol, Anatrace, Maumee, OH), methyl-β-cyclodextrin (MBCD, Sigma-Aldrich), MG132 (Merck Chemicals Ltd, Darmstadt, Germany), N-ethylmaleamide (NEM, Thermo Fisher Scientific), propidium iodide (Sigma-Aldrich), puromycin (Invivogen), SILAC Lys-8-Arg-10 kit (282986444, Silantes GmbH, Munich, Germany), sodium-meta-periodate (Sigma-Aldrich) and ZA (zaragozic acid A, Santa Cruz Biotechnology).

    Techniques: Isolation, Fractionation, Western Blot, Quantitative RT-PCR, Derivative Assay, Staining

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1004390

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

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

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

    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: Cells were incubated in 0.5 mg/mL filipin (stock diluted in PBS) in the dark, rinsed and mounted with ProLong Gold (Molecular Probes).

    Techniques: Labeling, Staining

    Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), filipin III (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m

    Journal: Cell Death & Disease

    Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity

    doi: 10.1038/cddis.2014.459

    Figure Lengend Snippet: Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), filipin III (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m

    Article Snippet: The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components.

    Techniques: Transduction, Labeling, Confocal Microscopy, Purification, Incubation

    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

    IL-36 signaling modulates cholesterol metabolism in Mtb -infected macrophage. ( A ) Changes in total cholesterol, free cholesterol and cholesteryl ester in cell lysates from scramble and IL36R KD THP-1 macrophages upon Mtb infection. ( B ) Immunofluorescence staining with Filipin III in non-infected (N/I), 24 h and 48 h Mtb -infected scramble versus IL36R KD THP-1 macrophages (40× objective magnification). ( C ) mRNA expression of CD36 , LDLR , SRA in Mtb -infected scramble versus IL36R KD cells. ( D ) Cholesterol efflux upon Mtb infection or LXR stimulation in scramble versus IL36R KD THP-1 macrophages with or without LXR inhibitors. ( E ) SREBP2 protein expression in Mtb -infected scramble and IL-36 deficient cells. ( F ) mRNA expression of several cholesterol synthesis genes in Mtb -infected macrophages at 30 h with or without 27HC, 25HC and betulin stimulation. ( A , C , D , F ) Data pooled from three independent experiments are shown. Data are shown as mean ± SD. ( B and E ) Data from one representative experiment of at least two independent experiments are shown. P values: ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Journal: Scientific Reports

    Article Title: IL-36/LXR axis modulates cholesterol metabolism and immune defense to Mycobacterium tuberculosis

    doi: 10.1038/s41598-018-19476-x

    Figure Lengend Snippet: IL-36 signaling modulates cholesterol metabolism in Mtb -infected macrophage. ( A ) Changes in total cholesterol, free cholesterol and cholesteryl ester in cell lysates from scramble and IL36R KD THP-1 macrophages upon Mtb infection. ( B ) Immunofluorescence staining with Filipin III in non-infected (N/I), 24 h and 48 h Mtb -infected scramble versus IL36R KD THP-1 macrophages (40× objective magnification). ( C ) mRNA expression of CD36 , LDLR , SRA in Mtb -infected scramble versus IL36R KD cells. ( D ) Cholesterol efflux upon Mtb infection or LXR stimulation in scramble versus IL36R KD THP-1 macrophages with or without LXR inhibitors. ( E ) SREBP2 protein expression in Mtb -infected scramble and IL-36 deficient cells. ( F ) mRNA expression of several cholesterol synthesis genes in Mtb -infected macrophages at 30 h with or without 27HC, 25HC and betulin stimulation. ( A , C , D , F ) Data pooled from three independent experiments are shown. Data are shown as mean ± SD. ( B and E ) Data from one representative experiment of at least two independent experiments are shown. P values: ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Article Snippet: To stain with Filipin III (Biomol), stock solution was diluted to 1:100 with PBS/BSA 3% and incubated for 2 h at RT in the dark.

    Techniques: Infection, Immunofluorescence, Staining, 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

    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

    ASM inhibitors induce melanoma-specific cell death by inhibiting intracellular cholesterol transport. ( A ) fluorescence microscopy of intracellular cholesterol localisation following knockdown of SMPD1 (ASM1) as detected by Filipin-III staining (arrows show accumulated cholesterol); ( B ) the viability of UACC 903 and 1205 Lu melanoma cells following knockdown of SMPD1; ( C ) IC 50 values of various melanoma cell lines and FF2441 fibroblasts treated with ASM inhibitors. Dose–response curves were drawn in OriginPro (OriginLab) using Levenberg Marquardt algorithm; ( D ) viability of UACC 903 and FF2441 cells following 24 h of treatment with increasing concentrations of ASM inhibitors; ( E ) viability of melanoma cells treated with various ASM inhibitors in the absence or presence of v-ATPase inhibitor, Bafilomycin-A1; ( F ) intracellular cholesterol localisation following leelamine, U18666A or ASM inhibitor treatments as detected by Filipin-III staining. (lower) Co-localisation of RFP-tagged lysosomal LAMP1 protein with cholesterol; ( G ) LDL treatment protects UACC 903 cells from ASM inhibitor-mediated cell death; ( H ) Venn diagram showing number of significantly altered genes identified by RNA-sequencing of UACC 903 cells treated with ASM inhibitors (left); and enrichment analysis of significantly altered 177 genes; ( I ) expression level of cholesterol synthesis genes following ASM treatment as detected by RNA-sequencing; ( J ) cholesterol synthesis pathway. White highlighted genes were identified as significantly deregulated by RNA-sequencing; ( K ) distribution of log-2-fold change in expression levels of cholesterol synthesis genes identified by microarray analysis following treatment of MCF7 cells with ASM inhibitors.

    Journal: British Journal of Cancer

    Article Title: Modulating cancer cell survival by targeting intracellular cholesterol transport

    doi: 10.1038/bjc.2017.200

    Figure Lengend Snippet: ASM inhibitors induce melanoma-specific cell death by inhibiting intracellular cholesterol transport. ( A ) fluorescence microscopy of intracellular cholesterol localisation following knockdown of SMPD1 (ASM1) as detected by Filipin-III staining (arrows show accumulated cholesterol); ( B ) the viability of UACC 903 and 1205 Lu melanoma cells following knockdown of SMPD1; ( C ) IC 50 values of various melanoma cell lines and FF2441 fibroblasts treated with ASM inhibitors. Dose–response curves were drawn in OriginPro (OriginLab) using Levenberg Marquardt algorithm; ( D ) viability of UACC 903 and FF2441 cells following 24 h of treatment with increasing concentrations of ASM inhibitors; ( E ) viability of melanoma cells treated with various ASM inhibitors in the absence or presence of v-ATPase inhibitor, Bafilomycin-A1; ( F ) intracellular cholesterol localisation following leelamine, U18666A or ASM inhibitor treatments as detected by Filipin-III staining. (lower) Co-localisation of RFP-tagged lysosomal LAMP1 protein with cholesterol; ( G ) LDL treatment protects UACC 903 cells from ASM inhibitor-mediated cell death; ( H ) Venn diagram showing number of significantly altered genes identified by RNA-sequencing of UACC 903 cells treated with ASM inhibitors (left); and enrichment analysis of significantly altered 177 genes; ( I ) expression level of cholesterol synthesis genes following ASM treatment as detected by RNA-sequencing; ( J ) cholesterol synthesis pathway. White highlighted genes were identified as significantly deregulated by RNA-sequencing; ( K ) distribution of log-2-fold change in expression levels of cholesterol synthesis genes identified by microarray analysis following treatment of MCF7 cells with ASM inhibitors.

    Article Snippet: Lysosomal/late endosomal localisation of cholesterol was assessed by co-localisation of LAMP1-RFP and Filipin-III signals using iVision software (BioVision Technologies, Chester Springs, PA, USA).

    Techniques: Fluorescence, Microscopy, Staining, RNA Sequencing Assay, Expressing, Microarray

    Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) Filipin staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

    doi: 10.3390/ijms20205185

    Figure Lengend Snippet: Correction of the cholesterol accumulation phenotype in a complementation test using different NPC1 variants. ( a ) Sanger sequencing results show the insertion of the desired point mutations into the vector containing NPC1 cDNA. ( b ) Filipin staining. WT NPC1 and different variants were investigated for cholesterol clearance in CHO Npc1 cells 12 h post transfection. ( c ) Quantification of the positive cholesterol signal. Values are shown as mean ± SD. Asterisks indicate statistical significance: ** p

    Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

    Techniques: Sequencing, Plasmid Preparation, Staining, Transfection

    Reduced cholesterol clearance in NPC1 -deficient Chinese hamster ovary (CHO) cells expressing variant p.Ile1061Thr NPC1 . CHO NPC1 cells develop a phenotype of cholesterol accumulation in lysosome-like storage organelles (LSOs). This phenotype can be corrected by gene transfer of wild type NPC1 cDNA using liposome-mediated cell transfection. ( a ) Fluorescence microscopic images. The blue staining visualizes cellular filipin-bound cholesterol. The arrowheads indicate LSOs. The green signal shows positively transfected cells that could be used for filipin quantification by either indicating GFP signal (control vector transfection) or NPC1-GFP fusion protein signal. ( b ) Quantification of the positive cholesterol signal. The y-axis shows the relative fluorescence intensity (RFI) signal as arbitrary units. Values are shown as mean ± SD. Asterisks indicate statistical significance: *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

    doi: 10.3390/ijms20205185

    Figure Lengend Snippet: Reduced cholesterol clearance in NPC1 -deficient Chinese hamster ovary (CHO) cells expressing variant p.Ile1061Thr NPC1 . CHO NPC1 cells develop a phenotype of cholesterol accumulation in lysosome-like storage organelles (LSOs). This phenotype can be corrected by gene transfer of wild type NPC1 cDNA using liposome-mediated cell transfection. ( a ) Fluorescence microscopic images. The blue staining visualizes cellular filipin-bound cholesterol. The arrowheads indicate LSOs. The green signal shows positively transfected cells that could be used for filipin quantification by either indicating GFP signal (control vector transfection) or NPC1-GFP fusion protein signal. ( b ) Quantification of the positive cholesterol signal. The y-axis shows the relative fluorescence intensity (RFI) signal as arbitrary units. Values are shown as mean ± SD. Asterisks indicate statistical significance: *** p

    Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

    Techniques: Expressing, Variant Assay, Transfection, Fluorescence, Staining, Plasmid Preparation

    Cholesterol elimination by the p.Ile1061Thr variant can be assisted by 25-hydroxycholesterol. The black bars indicate the filipin signal of untreated or 5 µM 25-hydroxycholesterol treated CHO NPC1 cells transfected with either pCMV6 control vector or WT NPC1-GFP vector 24 h post-transfection. Values are shown as mean ± SD. Asterisks indicate statistical significance: * p

    Journal: International Journal of Molecular Sciences

    Article Title: Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test

    doi: 10.3390/ijms20205185

    Figure Lengend Snippet: Cholesterol elimination by the p.Ile1061Thr variant can be assisted by 25-hydroxycholesterol. The black bars indicate the filipin signal of untreated or 5 µM 25-hydroxycholesterol treated CHO NPC1 cells transfected with either pCMV6 control vector or WT NPC1-GFP vector 24 h post-transfection. Values are shown as mean ± SD. Asterisks indicate statistical significance: * p

    Article Snippet: Prior to transfection with Lipofectamin 2000 the cells were allowed to adhere for 24 h. At 12 and 24 h after the transfection, the CHO cells were fixed using 4% PFA in PBS at pH 7.4 for 15 min followed by washing with PBS and incubation with filipin solution (0.5 mg/mL in PBS; Polysciences, Warrington, PA, USA) at room temperature in the dark for 45 min. After extended washing in PBS in a dark chamber, the coverslips were mounted with Mowiol mounting medium (Mowiol, #81831, Sigma) and stored at 4 °C until the analysis.

    Techniques: Variant Assay, Transfection, Plasmid Preparation

    Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p

    Journal: Redox Biology

    Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    doi: 10.1016/j.redox.2017.02.024

    Figure Lengend Snippet: Npc1 -/- mice liver and NPC cells show increased MLN64 expression. (A) To detect protein expression 30 μg of protein from liver homogenates were subjected to SDS-PAGE and western blotting for MLN64. ε-COP was used as a loading control. The Figure shows a western blot representative image, n=5. (B) Western blots bands intensity quantification. (C) Real time PCR analysis of MLN64 mRNA from mouse liver, n=4. (D) 30 μg of protein of CHO-WT and CHO-NPC cell extracts were analyzed by western blot against MLN64 and ε-COP. A representative image is shown, n=3. (E) Western blots bands intensity quantification. (F) MLN64 immunofluorescence analysis (Top). CHO-WT and CHO-NPC cells were immunostained with an anti-MLN64 antibody. Filipin staining (Bottom). CHO-WT and CHO-NPC cells were fixed, and cholesterol accumulation was detected by filipin staining. Scale bar: 20 µm (G) Graph shows quantifications of fluorescence. * Indicates statistically significant differences (p

    Article Snippet: Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

    Techniques: Mouse Assay, Expressing, SDS Page, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence

    MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p

    Journal: Redox Biology

    Article Title: MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    doi: 10.1016/j.redox.2017.02.024

    Figure Lengend Snippet: MLN64 overexpression increases mitochondrial cholesterol and decreases mitochondrial glutathione and ATPase activity in cultured hepatocytes (A) Immunofluorescence and filipin staining analysis of Ad.E1Δ and Ad.MLN64 HepG2 cells stained with anti-MLN64 (MLN64, red), anti-cytochrome c (Cyt-C, green) and filipin (cyan) 48 h after infection. Colocalization of Cyt-C and filipin indicating of mitochondrial cholesterol is depicted (white). Merge and zoom of the merge. Scale bar: 20 µm. (B) The graph shows the Pearson’s coefficient that measures the percentage of colocalization of filipin and Cyt-C. The photographs shown are representative confocal images, (n=3). (C) Mitochondrial glutathione content. Mitochondria were isolated by Percoll density ultracentrifugation from Ad.E1Δ and Ad.MLN64 HepG2 cells 48 h after infection. Glutathione in mitochondria was determined as described in the Materials and Methods section, (n=4). (D) ATPase activity in mitochondrial fraction from HepG2 cells (n=4) was quantified by measuring the hydrolysis rate of ATP. *Indicates statistically significant differences (p

    Article Snippet: Cells were incubated for 2 h with 25 μg/ml filipin, rinsed with PBS, followed by incubation with primary antibodies, mouse anti-cytochrome C antibody (1:200, ab13575, Abcam) and rabbit anti-MLN64 polyclonal antibody (1:500), rinsed with PBS, and incubated for 2 h with secondary antibodies (1:1000).

    Techniques: Over Expression, Activity Assay, Cell Culture, Immunofluorescence, Staining, Infection, Isolation

    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

    IGF2 induces lipid droplet formation but has no influence on lipid droplet size. (A) Representative LD540 fluorescence staining for lipid droplets (LD, yellow droplets) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were stained with DAPI (blue) (scale bar: 20 μm) (upper panel). Five randomly selected pictures per sample were collected and analyzed with ImageJ. Counted LDs were normalized to the nuclei count (lower panel, left ). Total LD area was normalized to the nuclei count (lower panel, middle ). Total LD size was estimated as the mean area of all counted LDs (lower panel, right ) (co, n = 10; Igf2, n = 11, independent two-sample t -test). (B) Representative filipin fluorescence staining for free cholesterol (black droplets) in Igf2 and co animals (co, n = 10; Igf2, n = 11, arrows designate examples for free cholesterol). (C) Levels of Hmgcr mRNA, real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA and co. All results are presented as mean ± SEM.

    Journal: Frontiers in Physiology

    Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation

    doi: 10.3389/fphys.2016.00147

    Figure Lengend Snippet: IGF2 induces lipid droplet formation but has no influence on lipid droplet size. (A) Representative LD540 fluorescence staining for lipid droplets (LD, yellow droplets) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were stained with DAPI (blue) (scale bar: 20 μm) (upper panel). Five randomly selected pictures per sample were collected and analyzed with ImageJ. Counted LDs were normalized to the nuclei count (lower panel, left ). Total LD area was normalized to the nuclei count (lower panel, middle ). Total LD size was estimated as the mean area of all counted LDs (lower panel, right ) (co, n = 10; Igf2, n = 11, independent two-sample t -test). (B) Representative filipin fluorescence staining for free cholesterol (black droplets) in Igf2 and co animals (co, n = 10; Igf2, n = 11, arrows designate examples for free cholesterol). (C) Levels of Hmgcr mRNA, real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA and co. All results are presented as mean ± SEM.

    Article Snippet: Slides were embedded in the water based FluoroSafe™ mounting medium and visualized with an inverse fluorescence microscope at 558/583 (excitation/emission) for LD540 and at 359/461 (excitation/emission) for DAPI and filipin (Axio Observer, Zeiss, Feldbach, Swiss).

    Techniques: Fluorescence, Staining, Injection, Plasmid Preparation, Quantitative RT-PCR

    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

    Quercetin and lipid accumulation in RAW264.7 cells induced by ox-LDL. (A) RAW264.7 cells were treated with quercetin and ox-LDL and the lipid accumulation was measured by oil red staining. (B) RAW264.7 cells were treated with quercetin and ox-LDL and the intracellular free cholesterol was measured using Filipin staining. (C) RAW264.7 cells were treated with quercetin and ox-LDL and the total cellular cholesterol was measured. Data are presented as the mean ± standard deviation. *P

    Journal: Molecular Medicine Reports

    Article Title: Quercetin protects against ox-LDL-induced injury via regulation of ABCAl, LXR-α and PCSK9 in RAW264.7 macrophages

    doi: 10.3892/mmr.2018.9048

    Figure Lengend Snippet: Quercetin and lipid accumulation in RAW264.7 cells induced by ox-LDL. (A) RAW264.7 cells were treated with quercetin and ox-LDL and the lipid accumulation was measured by oil red staining. (B) RAW264.7 cells were treated with quercetin and ox-LDL and the intracellular free cholesterol was measured using Filipin staining. (C) RAW264.7 cells were treated with quercetin and ox-LDL and the total cellular cholesterol was measured. Data are presented as the mean ± standard deviation. *P

    Article Snippet: A Filipin staining kit was purchased from Shanghai Genmed (Shanghai, China), and DAPI staining solution was purchased from Shanghai Beyotime.

    Techniques: Staining, Standard Deviation

    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

    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, Filipin staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p

    Journal: Scientific Reports

    Article Title: Endoplasmic Reticulum Stress May Play a Pivotal Role in Lipid Metabolic Disorders in a Novel Mouse Model of Subclinical Hypothyroidism

    doi: 10.1038/srep31381

    Figure Lengend Snippet: Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, Filipin staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p

    Article Snippet: Frozen sections of the liver (5 μm) were stained using a filipin fluorescence staining kit (Genmed Scientifics Inc., USA), according to the manufacturer’s recommended protocol.

    Techniques: Mouse Assay, Cholesterol Assay, Staining, Real-time Polymerase Chain Reaction

    Treatment with 4-PBA alleviated lipid metabolic disorders in SCH mice. (A) Levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in the serum of all mice were determined after 4-PBA was administered for 4 weeks (n = 6–12). (B) Hepatic TC contents were detected using a cholesterol assay kit after 4-PBA was administered for 4 weeks (n = 6–12). (C) Hepatic TG contents were detected using a triglyceride assay kit after 4-PBA was administered for 4 weeks (n = 6–13). (D) Filipin staining was used to measure hepatic cholesterol deposition after 4-PBA was administered for 4 weeks (n = 3). Original magnification: 200×. (E) Oil red O staining was used to measure hepatic TG accumulation after 4-PBA was administered for 4 weeks (n = 3). Original magnification: 200×. (F) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 18th week (n = 4–6). The data are presented as the mean ± SD. *p

    Journal: Scientific Reports

    Article Title: Endoplasmic Reticulum Stress May Play a Pivotal Role in Lipid Metabolic Disorders in a Novel Mouse Model of Subclinical Hypothyroidism

    doi: 10.1038/srep31381

    Figure Lengend Snippet: Treatment with 4-PBA alleviated lipid metabolic disorders in SCH mice. (A) Levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in the serum of all mice were determined after 4-PBA was administered for 4 weeks (n = 6–12). (B) Hepatic TC contents were detected using a cholesterol assay kit after 4-PBA was administered for 4 weeks (n = 6–12). (C) Hepatic TG contents were detected using a triglyceride assay kit after 4-PBA was administered for 4 weeks (n = 6–13). (D) Filipin staining was used to measure hepatic cholesterol deposition after 4-PBA was administered for 4 weeks (n = 3). Original magnification: 200×. (E) Oil red O staining was used to measure hepatic TG accumulation after 4-PBA was administered for 4 weeks (n = 3). Original magnification: 200×. (F) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 18th week (n = 4–6). The data are presented as the mean ± SD. *p

    Article Snippet: Frozen sections of the liver (5 μm) were stained using a filipin fluorescence staining kit (Genmed Scientifics Inc., USA), according to the manufacturer’s recommended protocol.

    Techniques: Mouse Assay, Cholesterol Assay, Staining, Real-time Polymerase Chain Reaction