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  • 99
    Millipore filipin
    Effect of endocytic-inhibitors on accumulation of Lipodox ® and cell viability in HepG2 cells. Notes: Cells were either untreated or pretreated under separate conditions with various inhibitors. ( A ) NaN 3 as ATPase-inhibitor, ( B ) CPZ, ( C ) wortmannin, ( D ) <t>filipin</t> for 30 minutes at 37°C prior to adding Lipodox ® (50 µM). The fluorescence signal was measured following incubation for 3 hours at 37°C in all cases. ( E ) HepG2 cells for viability assay were cultured for 24 hours after treatment, and viable cell yields were determined by the Trypan Blue exclusion method. Data represent mean ± SD (n=3). * P
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2040 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical filipin iii
    Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), <t>filipin</t> <t>III</t> (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml −1 PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells
    Filipin Iii, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 171 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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    Polysciences inc filipin
    Effects of PKC activation on the NPC1 phenotype. NPC1 CHO cells (B and E) were treated with 100µM DCP-LA (C), 10µM DHA (D), or 100µM diazoxide (E) and cholesterol storage was quantified by <t>filipin</t> fluorescence. (A) Wt CHO cells. Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. *, **, and *** denote statistically significant differences between treated and untreated cells with P
    Filipin, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Santa Cruz Biotechnology filipin iii
    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) <t>Filipin</t> <t>III</t> 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).
    Filipin Iii, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore filipin dye
    Effect of methyl-β-cyclodextrin (MβCD) on reduction of cholesterol accumulation and lysosome size in NPC fibroblasts determined by Lysotracker-red staining, <t>filipin</t> cholesterol staining and Amplex-red cholesterol assays. (A) Images of
    Filipin Dye, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Merck & Co filipin iii
    Effect of methyl-β-cyclodextrin (MβCD) on reduction of cholesterol accumulation and lysosome size in NPC fibroblasts determined by Lysotracker-red staining, <t>filipin</t> cholesterol staining and Amplex-red cholesterol assays. (A) Images of
    Filipin Iii, supplied by Merck & Co, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    AG Scientific filipin complex
    Effect of methyl-β-cyclodextrin (MβCD) on reduction of cholesterol accumulation and lysosome size in NPC fibroblasts determined by Lysotracker-red staining, <t>filipin</t> cholesterol staining and Amplex-red cholesterol assays. (A) Images of
    Filipin Complex, supplied by AG Scientific, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    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: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Omega Optical 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 Omega Optical, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore prepared filipin iii
    GP73 regulates the transcriptional activity of SREBPs and lipogenesis. ( a ) Immunoblotting analysis of SREBPs activation in HepG2 cells transfected with Flag-GP73 at the indicated doses. α-Tubulin was used as equal loading control. ( b , c ) SREBP-1 promoter activity in HepG2 ( b ) or HL7702 ( c ) cells transfected with Flag-vector or Flag-GP73 under conditions of sterol depletion or repletion. The luciferase activity was measured 36 hrs post transfection. The value was normalized with the corresponding transfection efficiency. ( d , f , h ) QRT-PCR analysis of HMGR ( d ), FASN2 ( f ), and ACC1 ( h ) mRNA abundance in HepG2 cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( e , g , i ) QRT-PCR analysis of HMGR ( e ), FASN2 ( g ), and ACC1 ( i ) mRNA abundance in 293T cells transfected with Flag-vector or Flag-GP73 for 24 hrs. ( j ) Fluorescence microscopy of <t>Filipin</t> staining in HepG2 cells transfected with Flag-GP73. Cells were collected at indicated hrs post transfection. ( k , l ) Amplex Red cholesterol assay of cellular cholesterol concentrations in HepG2 ( k ) or HL7702 ( l ) cells transfected with Flag-vector or Flag-GP73. Cells were collected at indicated hrs post transfection. Values were normalized to total cell proteins from control cells transfected with Flag-vector. Cell-based studies were performed at least <t>three</t> independent times with comparable results. Data represent mean ± SEM. Student’s t test was used for statistical analysis: **p
    Prepared Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore filipin sigma
    VDR silencing (shVDR) in HepG2 cells demonstrates that VDR is a potential negative regulator of FETUB. A. Representative PCR performed on gDNA from shVDR clones, viral controls (EB8 and EB10) and HepG2 cells (-ve control) using backbone specific primers to demonstrate successful integration. Concentration and gDNA quality were assessed a Gapdh PCR. Integration PCRs were performed for each independent shRNA delivery (n = 3). B. Western immunoblot analyses under reduced conditions in 20ug of whole cell lysates from shVDR clones (shVDR) and controls (CTR). Antibodies specific for VDR, FETUB, RXRα and ERK were utilized to analyze their endogenous expression, with results normalized to total protein. Gels are representative of n = 3 experiments for 3 independent viral deliveries performed. C. Representative western immunoblot analyses for RXRα and ERK of 2DE with 100ug of whole cell lysates from shVDR clones (shVDR542) and controls (EB8). Arrows highlight pI shifts observed for RXRα and ERK1 p44 and ERK2 p42. D. Representative western immunoblot analyses of endogenous FETUB (FETUBendog) under reduced conditions in 20ug of whole cell lysates from shVDR clones and controls (shEB8 or HepG2) following a 1hr treatment with increasing concentrations of <t>filipin</t> (1–10 ug/ml) or CPZH (5-10ug/ml), followed by 24 h in maintenance medium. Results are representative of 4 independent shVDR clones and are normalized to total protein (n = 3).
    Filipin Sigma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore lr disruptors filipin
    Effects of Oxo on ASMase and ADP-ribosylcyclase activity in CAMs in the absence or presence of LR disruptors. A : summarized ASMase activity of CAMs treated with Oxo (80 μM, 15 min) alone or with 20-min pretreatment of MCD (100 μM), <t>filipin</t>
    Lr Disruptors Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    FUJIFILM 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 FUJIFILM, used in various techniques. Bioz Stars score: 86/100, based on 3 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: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lipid raft inhibitor filipin iii
    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
    Lipid Raft Inhibitor Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sterol binding compound filipin
    Specific tissues and membrane regions retain sterols preferentially. ( A-T ) Second instar larval tissues (represented diagrammatically in A,F,K,P) stained with <t>filipin.</t> The CNS (A-E), salivary glands (F-J), fat bodies (K-O) and midguts (P-T) from wild-type
    Sterol Binding Compound Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Abcam filipin staining
    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by <t>Filipin</t> in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)
    Filipin Staining, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical filipin stain
    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by <t>Filipin</t> in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)
    Filipin Stain, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical filipin staining
    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by <t>Filipin</t> in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)
    Filipin Staining, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genmed filipin staining
    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by <t>Filipin</t> in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)
    Filipin Staining, supplied by Genmed, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of endocytic-inhibitors on accumulation of Lipodox ® and cell viability in HepG2 cells. Notes: Cells were either untreated or pretreated under separate conditions with various inhibitors. ( A ) NaN 3 as ATPase-inhibitor, ( B ) CPZ, ( C ) wortmannin, ( D ) filipin for 30 minutes at 37°C prior to adding Lipodox ® (50 µM). The fluorescence signal was measured following incubation for 3 hours at 37°C in all cases. ( E ) HepG2 cells for viability assay were cultured for 24 hours after treatment, and viable cell yields were determined by the Trypan Blue exclusion method. Data represent mean ± SD (n=3). * P

    Journal: International Journal of Nanomedicine

    Article Title: Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells

    doi: 10.2147/IJN.S188791

    Figure Lengend Snippet: Effect of endocytic-inhibitors on accumulation of Lipodox ® and cell viability in HepG2 cells. Notes: Cells were either untreated or pretreated under separate conditions with various inhibitors. ( A ) NaN 3 as ATPase-inhibitor, ( B ) CPZ, ( C ) wortmannin, ( D ) filipin for 30 minutes at 37°C prior to adding Lipodox ® (50 µM). The fluorescence signal was measured following incubation for 3 hours at 37°C in all cases. ( E ) HepG2 cells for viability assay were cultured for 24 hours after treatment, and viable cell yields were determined by the Trypan Blue exclusion method. Data represent mean ± SD (n=3). * P

    Article Snippet: In the inhibition experiments, different inhibitors for each endocytosis pathway were used and cells were pre-treated with them for 30 minutes prior to mEHT treatment, including 200 µg/mL NaN3 (Sigma S2002, ATPase-inhibitor, Sigma-Aldrich Co., St Louis, MO, USA), 4 µg/mL filipin (Sigma F4767, caveolae-mediated pathway), 0.1 µM wortmannin (Sigma W3144, macropinocytosis), and 7 µg/mL chlorpromazine (CPZ) (Sigma C8138, clathrin-mediated endocytosis pathway).

    Techniques: Fluorescence, Incubation, Viability Assay, Cell Culture

    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

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

    Journal: Autophagy

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

    doi: 10.4161/15548627.2014.984272

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

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

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

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

    Journal: The Journal of Neuroscience

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

    doi: 10.1523/JNEUROSCI.2942-18.2019

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

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

    Techniques: shRNA, Plasmid Preparation, Transfection

    Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), filipin III (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml −1 PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells

    Journal: Bioengineering & Translational Medicine

    Article Title: Uptake and function of membrane‐destabilizing cationic nanogels for intracellular drug delivery. Uptake and function of membrane‐destabilizing cationic nanogels for intracellular drug delivery

    doi: 10.1002/btm2.10120

    Figure Lengend Snippet: Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), filipin III (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml −1 PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells

    Article Snippet: Filipin III was purchased from Cayman Chemical (Ann Arbor, MI).

    Techniques: Staining, Cytometry, Incubation, Generated

    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

    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

    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

    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

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

    Journal: Journal of biomolecular screening

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

    doi: 10.1177/1087057113501197

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

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

    Techniques: Staining

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

    Journal: Autophagy

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

    doi: 10.1080/15548627.2017.1329081

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

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

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

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

    Journal: Autophagy

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

    doi: 10.1080/15548627.2017.1329081

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

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

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

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

    Journal: Autophagy

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

    doi: 10.1080/15548627.2017.1329081

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

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

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

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

    Journal: Autophagy

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

    doi: 10.1080/15548627.2017.1329081

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

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

    Techniques: Staining, Western Blot, Expressing

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.278754

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

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

    Techniques: Staining

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

    Journal: Assay and Drug Development Technologies

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

    doi: 10.1089/adt.2017.774

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

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

    Techniques: Staining, Ethidium Homodimer Assay

    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

    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

    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

    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

    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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0211187

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

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

    Techniques: Staining

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0211187

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

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

    Techniques: Incubation, Staining

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0211187

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

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

    Techniques: Staining, Fluorescence, Incubation

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.06111-11

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

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

    Techniques: Activation Assay

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

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

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

    doi: 10.1016/j.jsbmb.2018.04.009

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

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

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

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

    Journal: Journal of neuropathology and experimental neurology

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

    doi: 10.1097/NEN.0b013e3181815994

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

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

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

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

    Journal: Journal of neuropathology and experimental neurology

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

    doi: 10.1097/NEN.0b013e3181815994

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0154366

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

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

    Techniques: Marker, Plasmid Preparation, Expressing

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

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.113.204958

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

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

    Techniques: Inhibition, Light Microscopy, Staining

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

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.113.204958

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

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

    Techniques: Staining

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

    Journal: Microbial Cell Factories

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

    doi: 10.1186/s12934-015-0307-4

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

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

    Techniques:

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

    Journal: Microbial Cell Factories

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

    doi: 10.1186/s12934-015-0307-4

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

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

    Techniques: Purification, Microdilution Assay

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

    Journal: Microbial Cell Factories

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

    doi: 10.1186/s12934-015-0307-4

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

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

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis

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

    Journal: Microbial Cell Factories

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

    doi: 10.1186/s12934-015-0307-4

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

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

    Techniques: Generated, Thin Layer Chromatography

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

    Journal: Oncotarget

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

    doi:

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

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

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

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

    Journal: Molecular Vision

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

    doi:

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

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

    Techniques: Cell Culture, Staining, Positive Control

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0026289

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

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

    Techniques: Labeling, Staining

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

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

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

    doi: 10.1073/pnas.0808720105

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1006257

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

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

    Techniques: Infection, Activation Assay, Staining, Confocal Microscopy

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0005193

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0005193

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

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

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

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

    Journal: Cell Communication and Signaling : CCS

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

    doi: 10.1186/s12964-014-0057-7

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

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

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

    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

    Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by Filipin in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)

    Journal: Cell Death & Disease

    Article Title: Specific antibody binding to the APP672–699 region shifts APP processing from α- to β-cleavage

    doi: 10.1038/cddis.2014.336

    Figure Lengend Snippet: Colocalization of human wild-type APP with cholesterol in CHO/APP wt cells after mAb ED-C99 treatment. ( a ) CHO/APP wt cells were plated to 8-well slide chamber and then, after overnight incubation, the cells were treated with mAb ED-C99 or IgG 1 isotype control for 2 h. These cells were stained by Filipin in strict accordance with the manufacturer's instructions of the cholesterol assay kit. ( b ) After Filipin staining and washing, some of these cells were permeabilized with 0.05% Triton X-100 for 5 min, washed, and stained with rabbit anti-APP-C-terminal antibody overnight at 4°C. Alexa Fluor 594 Donkey anti-rabbit IgG was used to detect APP signals. Confocal images were taken by Olympus fluoview FV1000 laser scanning confocal microscope (Tokyo, Japan)

    Article Snippet: Filipin staining After treatment, the cells were fixed and Filipin staining was performed following the manufacturer's instructions of the cholesterol assay kit (AbCam).

    Techniques: Incubation, Staining, Cholesterol Assay, Microscopy