filipin stain Search Results


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  • 99
    Millipore filipin stain
    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 <t>filipin</t> 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).
    Filipin Stain, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cayman Chemical filipin stain
    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 <t>filipin</t> 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).
    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
    https://www.bioz.com/result/filipin stain/product/Cayman Chemical
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    92
    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: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Genmed filipin fluorescence staining kit
    Hyperlipidemia and hepatic lipid metabolic disorders in the SCH mice. Male C57/BL6 mice were administered methimazole (MMI, 0.08 mg/kg·BW·d, SCH group, n = 6–8) or a corresponding volume of vehicle (control group, n = 6–8) for 12 weeks or 16 weeks. (A) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 12th week. (B) Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG) in the serum of all mice were determined in the 16th week. (C) Liver TC contents were measured using a cholesterol assay kit in the 12th week and the 16th week. (D) i, <t>Filipin</t> staining was used to measure liver cholesterol deposition in the 12th week. ii, Filipin staining was used to measure liver cholesterol deposition in the 16th week. Original magnification: 200×. (E) Liver TG contents were measured using a triglyceride assay kit in the 12th week and the 16th week. (F) i, Oil red O staining was used to measure liver TG accumulation in the 12th week. ii, Oil red O staining was used to measure liver TG accumulation in the 16th week. Original magnification: 200×. (G) Real-time PCR analysis of different lipid metabolism-related genes in the liver was performed in the 12th week. The data are presented as the mean ± SD. *p
    Filipin Fluorescence Staining Kit, supplied by Genmed, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    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: 90/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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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    93
    Genmed filipin staining a fc filipin fluorescent staining kit
    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 A Fc Filipin Fluorescent Staining Kit, supplied by Genmed, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore cholesterol staining filipin complex
    Distribution of cholesterol in membranes of normal human dermal fibroblasts NHDF (left) and melanoma cells A375 (right). Cholesterol was stained with <t>filipin</t> (green) and nucleus with PI (red). Overlay at the bottom. Pictures are representative results of more than three independent experiments.
    Cholesterol Staining Filipin Complex, supplied by Millipore, 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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    95
    Millipore filipin staining filipin dye
    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 <t>filipin</t> staining assay. Data represent mean ± SEM of 3 replicates. For this and all subsequent figures, * p
    Filipin Staining Filipin Dye, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beaverbio filipin staining cells
    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 <t>filipin</t> staining assay. Data represent mean ± SEM of 3 replicates. For this and all subsequent figures, * p
    Filipin Staining Cells, supplied by Beaverbio, 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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    99
    Millipore filipin staining cells
    ER stress alters cholesterol distribution but not cholesterol content. HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. A: Lipids were extracted, and free and esterified cholesterol were directly quantified by GC. Data show means ± SD from three experiments. CE, esterified cholesterol; FC, free cholesterol; TC, total cholesterol. B: Free cholesterol distribution was visualized by <t>Filipin</t> staining. Cholesterol distribution is altered by ER stress, although no quantitative alterations in FC and CE exist. Bar = 10 µm. Representative images from three independent experiments are shown.
    Filipin Staining Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Becton Dickinson direct quantitative filipin staining cells
    Overview of NP-C1 patient fibroblasts analyses. Strip plots show results of assays for the four phenotypic subgroups. Solid circles represent individual patient cell lines. Numbers adjacent to the circles represent patient number. The long-dashed and dashed horizontal lines represent the average control levels and standard error of means, respectively. Asterisks above the boxplots (2a, 2b, 2d, 2e) mark groups that differ significantly from controls. The groups were compared by one-way ANOVA test followed by post-hoc Tukey’s HSD test adjusted for unequal sample sizes. a NPC1 mRNA expression level, b NPC1 protein level by Western Blotting, c endosomal / lysosomal localisation of NPC1 protein. NPC1 vs. LAMP2 Object Pearson’s colocalization coefficient, d unesterified cholesterol / cholesterol ester ratio by MS/MS, e direct <t>filipin</t> staining of cellular unesterified cholesterol, f cholesterol esterification rate of LDL-derived cholesterol
    Direct Quantitative Filipin Staining Cells, supplied by Becton Dickinson, 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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    99
    Abcam filipin staining intracellular unesterified cholesterol
    Overview of NP-C1 patient fibroblasts analyses. Strip plots show results of assays for the four phenotypic subgroups. Solid circles represent individual patient cell lines. Numbers adjacent to the circles represent patient number. The long-dashed and dashed horizontal lines represent the average control levels and standard error of means, respectively. Asterisks above the boxplots (2a, 2b, 2d, 2e) mark groups that differ significantly from controls. The groups were compared by one-way ANOVA test followed by post-hoc Tukey’s HSD test adjusted for unequal sample sizes. a NPC1 mRNA expression level, b NPC1 protein level by Western Blotting, c endosomal / lysosomal localisation of NPC1 protein. NPC1 vs. LAMP2 Object Pearson’s colocalization coefficient, d unesterified cholesterol / cholesterol ester ratio by MS/MS, e direct <t>filipin</t> staining of cellular unesterified cholesterol, f cholesterol esterification rate of LDL-derived cholesterol
    Filipin Staining Intracellular Unesterified Cholesterol, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    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

    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

    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

    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

    ER stress alters cholesterol distribution but not cholesterol content. HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. A: Lipids were extracted, and free and esterified cholesterol were directly quantified by GC. Data show means ± SD from three experiments. CE, esterified cholesterol; FC, free cholesterol; TC, total cholesterol. B: Free cholesterol distribution was visualized by Filipin staining. Cholesterol distribution is altered by ER stress, although no quantitative alterations in FC and CE exist. Bar = 10 µm. Representative images from three independent experiments are shown.

    Journal: Journal of Lipid Research

    Article Title: Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells [S]

    doi: 10.1194/jlr.M043299

    Figure Lengend Snippet: ER stress alters cholesterol distribution but not cholesterol content. HepG2 cells were treated with 0.1 µM thapsigargin in media containing 10% LPDS for 24 h. A: Lipids were extracted, and free and esterified cholesterol were directly quantified by GC. Data show means ± SD from three experiments. CE, esterified cholesterol; FC, free cholesterol; TC, total cholesterol. B: Free cholesterol distribution was visualized by Filipin staining. Cholesterol distribution is altered by ER stress, although no quantitative alterations in FC and CE exist. Bar = 10 µm. Representative images from three independent experiments are shown.

    Article Snippet: Filipin staining Cells were fixed with 4% formaldehyde in PBS at 4°C for 30 min and stained with 50 µg/ml Filipin III (Sigma-Aldrich) diluted in PBS containing 10% LPDS at RT for 30 min.

    Techniques: Staining

    Overview of NP-C1 patient fibroblasts analyses. Strip plots show results of assays for the four phenotypic subgroups. Solid circles represent individual patient cell lines. Numbers adjacent to the circles represent patient number. The long-dashed and dashed horizontal lines represent the average control levels and standard error of means, respectively. Asterisks above the boxplots (2a, 2b, 2d, 2e) mark groups that differ significantly from controls. The groups were compared by one-way ANOVA test followed by post-hoc Tukey’s HSD test adjusted for unequal sample sizes. a NPC1 mRNA expression level, b NPC1 protein level by Western Blotting, c endosomal / lysosomal localisation of NPC1 protein. NPC1 vs. LAMP2 Object Pearson’s colocalization coefficient, d unesterified cholesterol / cholesterol ester ratio by MS/MS, e direct filipin staining of cellular unesterified cholesterol, f cholesterol esterification rate of LDL-derived cholesterol

    Journal: Orphanet Journal of Rare Diseases

    Article Title: Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

    doi: 10.1186/s13023-020-01360-5

    Figure Lengend Snippet: Overview of NP-C1 patient fibroblasts analyses. Strip plots show results of assays for the four phenotypic subgroups. Solid circles represent individual patient cell lines. Numbers adjacent to the circles represent patient number. The long-dashed and dashed horizontal lines represent the average control levels and standard error of means, respectively. Asterisks above the boxplots (2a, 2b, 2d, 2e) mark groups that differ significantly from controls. The groups were compared by one-way ANOVA test followed by post-hoc Tukey’s HSD test adjusted for unequal sample sizes. a NPC1 mRNA expression level, b NPC1 protein level by Western Blotting, c endosomal / lysosomal localisation of NPC1 protein. NPC1 vs. LAMP2 Object Pearson’s colocalization coefficient, d unesterified cholesterol / cholesterol ester ratio by MS/MS, e direct filipin staining of cellular unesterified cholesterol, f cholesterol esterification rate of LDL-derived cholesterol

    Article Snippet: Direct quantitative filipin staining Cells were seeded onto BD Falcon culture slides and cellular cholesterol accumulation was visualized and quantified after direct filipin staining [ ].

    Techniques: Stripping Membranes, Expressing, Western Blot, Tandem Mass Spectroscopy, Staining, Derivative Assay

    a NPC1 promoter haplotype variants and respective luciferase reporter activities in %. Reporter activity of pGL3 basic vector was 0.1 ± 0.1% of Haplotype 1 construct activity. Haplotype 3 is present in controls only. b Immunoreactive NPC1 protein in skin fibroblast lines (Western blotting). The cell line numbers and phenotypes are indicated on the top. Equal amount of protein (8 μg) was applied per line. Abnormal banding associated with p.Y276H is indicated by arrowheads. CCD data were used for the quantification. c The mutations are depicted using crystal structure of NPC1 protein 3JD8 [ 40 ] and the domains are color-coded according to Li [ 11 ]. A schematic of primary structure of the mature NPC1 protein is shown below the structure - domain color coding. NTD – N-terminal domain, TM – transmembrane domain, MLD - middle luminal domain, SSD – sterol sensing domain. The most severe mutations are indicated in bold font. Most of the mutations are in lumenal domains I and C (color-coded circles beside the mutation labels). d Representative images of human skin fibroblasts of a control and patients with selected forms of the disease. 1st column: direct filipin stained cultures. 2nd column: confocal microscopy images anti-NPC1 signal. 3rd column: merge of anti-LAMP2 (red) and anti-NPC1 (green) signal, 4th column: co-localization overlay maps. Values 0 - 1 of the pixels are displayed using lookup table LUT 0–1. All images were processed equally

    Journal: Orphanet Journal of Rare Diseases

    Article Title: Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

    doi: 10.1186/s13023-020-01360-5

    Figure Lengend Snippet: a NPC1 promoter haplotype variants and respective luciferase reporter activities in %. Reporter activity of pGL3 basic vector was 0.1 ± 0.1% of Haplotype 1 construct activity. Haplotype 3 is present in controls only. b Immunoreactive NPC1 protein in skin fibroblast lines (Western blotting). The cell line numbers and phenotypes are indicated on the top. Equal amount of protein (8 μg) was applied per line. Abnormal banding associated with p.Y276H is indicated by arrowheads. CCD data were used for the quantification. c The mutations are depicted using crystal structure of NPC1 protein 3JD8 [ 40 ] and the domains are color-coded according to Li [ 11 ]. A schematic of primary structure of the mature NPC1 protein is shown below the structure - domain color coding. NTD – N-terminal domain, TM – transmembrane domain, MLD - middle luminal domain, SSD – sterol sensing domain. The most severe mutations are indicated in bold font. Most of the mutations are in lumenal domains I and C (color-coded circles beside the mutation labels). d Representative images of human skin fibroblasts of a control and patients with selected forms of the disease. 1st column: direct filipin stained cultures. 2nd column: confocal microscopy images anti-NPC1 signal. 3rd column: merge of anti-LAMP2 (red) and anti-NPC1 (green) signal, 4th column: co-localization overlay maps. Values 0 - 1 of the pixels are displayed using lookup table LUT 0–1. All images were processed equally

    Article Snippet: Direct quantitative filipin staining Cells were seeded onto BD Falcon culture slides and cellular cholesterol accumulation was visualized and quantified after direct filipin staining [ ].

    Techniques: Luciferase, Activity Assay, Plasmid Preparation, Construct, Western Blot, Mutagenesis, Staining, Confocal Microscopy