Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: Selective recognition of cholesterol by GST-PFO. (A) Protein-lipid overlay assay. Indicated amounts of lipids were spotted on nitrocellulose membrane and incubated with 1 μg/ml GST-PFO. Immunoreactive spots were revealed by chemiluminescence. (B) 6-Carboxyfluorescein release from liposomes of indicated composition induced by 15 μg/ml GST-PFO. The data are expressed as percentage of the total amount of 6-carboxyfluorescein released from liposomes by 0.2% Triton X-100. (C) SPR sensograms of binding of 10 μg/ml GST-PFO or 20 μg/ml GST to liposomes of indicated lipid composition immobilized on the surface of sensor chip L1. Binding of GST-PFO to liposomes was carried out for 600 s after which the samples were washed for another 600 s. In (A) and (C) data represent one of three independent experiments; in (B) mean ± SEM from three experiments is shown. Chol – cholesterol; Cer – ceramide; SM – sphingomyelin.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Protein-lipid Overlay Assay (PLOA), Membrane, Incubation, Liposomes, Binding Assay

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: GST-PFO labels cholesterol deposits in NPC fibroblasts. (A-C) NPC fibroblasts were cultured in DMEM/10% FBS (A, A’) or in DMEM supplemented with delipidated 10% FBS for 72 h (B-C’) . Inset in (B’) shows an enlargement of a merged image of an area marked in (B, B’) . Colocalization of GST-PFO and filipin staining is in white. Colocalization of the labels is also seen in vesicles marked by arrows in (B, B’) . (C, C’) Omitting GST-PFO during labeling of permeabilized NPC cells yields only traces of non-specific staining with secondary antibodies (C) . In these conditions, filipin detects cholesterol accumulated in the cells (C’) . (D-D’) Fibroblasts from a healthy donor were cultured in DMEM/delipidated 10% FBS. Cells were fixed, permeabilized with 0.05% Triton X-100 and incubated with 5 μg/ml GST-PFO followed by secondary antibodies conjugated with Texas Red (left) or with 25 μg/ml filipin (right) to compare the staining patterns revealed by the two probes. Scale bar, 20 μm.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Cell Culture, Staining, Labeling, Incubation

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: GST-PFO labels vesicles containing DiI-LDL in NPC cells. NPC fibroblasts (A-A”) and healthy control fibroblasts (B-B”) were cultured in DMEM supplemented with delipidated 10% FBS for 72 h, incubated with 10 μg/ml DiI-LDL for 5 h and, after fixation and permeabilization, processed for GST-PFO labeling. (A, B) GST-PFO staining, (A’, B’) fluorescence of DiI-LDL, (A”, B”) merged images. Inset in (A” ) shows an enlargement of a marked area. Scale bar, 20 μm.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Control, Cell Culture, Incubation, Labeling, Staining, Fluorescence

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: Colocalization of GST-PFO with LAMP-1 and filipin in NPC fibroblasts. Cholesterol visualized by GST-PFO (red) is present in lysosomes identified by anti-LAMP-1 (green in A’-A” ) and filipin (blue in B’-B” ). (A”s1-s4) Merged images of optical sections of the cell shown in (A-A”) . The sections range from the basolateral (A”s1) to the apical (A”s4) part of the cell. (A”, B”) Merged images of (A-A’) and (B-B’) , respectively. Colocalization of GST-PFO and LAMP-1 is seen as yellow color in (A” and A”s1-s4) while pink color reflects colocalization of GST-PFO- and filipin-positive structures in (B”) . Scale bars, 20 μm.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques:

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: Quantitation of colocalization of GST-PFO-positive structures and other cellular components in NPC fibroblasts. (A) Pearson’s correlation coefficient, (B) overlap coefficient for co-immunostaining with GST-PFO and filipin or antibodies against LAMP1, LBPA, golgin-84, PDI, PMP70 or phalloidin used as an actin filament marker. TX, Triton X-100, DiT, digitonin. Data shown are mean ± SEM.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Quantitation Assay, Immunostaining, Marker

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: GST-PFO labeling of NPC cells colocalizes with LBPA. NPC (A-B”) and healthy control fibroblasts (C-C”) were fixed, permeabilized with 0.05% Triton X-100 (A-A”) or 0.05% digitonin (B-C”) and labeled with GST-PFO and anti-LBPA antibody. (A, B, C) GST-PFO labeling, (A’, B’, C’) LBPA localization, (A”, B”, C”) merged images. TX, Triton X-100, DiT, digitonin. Scale bar, 20 μm.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Labeling, Control

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: Negligible co-immunostaining of GST-PFO-positive structures and other organelles in NPC cells. (A-D) Distribution of GST-PFO-positive structures, (A’-D’) patterns of staining by antibodies directed against golgin-84 (A’) , PDI - an endoplasmic reticulum marker (B’) , anti-PMP70 – a peroxisomal marker (C’) and phalloidin labeling actin filaments (D’) . (A”-D”) merging of the corresponding pairs of images displays traces of the yellow color indicating different localization of GST-PFO-positive structures and the examined proteins. Scale bar, 20 μm.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Immunostaining, Staining, Marker, Labeling

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: Immunoelectron microscopy analysis of GST-PFO staining in NPC and healthy fibroblasts. Ultrathin sections of NPC cells (A, B) or control healthy fibroblasts (C) were subjected to post-embedding immunogold labeling with GST-PFO followed by anti-GST-biotin and anti-biotin-gold antibodies (6 nm) together with rabbit anti-LAMP-1 and anti-rabbit-gold antibodies (10 nm). GST-PFO decorates material accumulated in vesicles of NPC cells (arrows in A , B ) while LAMP-1 is visualized mainly at the borders of vesicles in NPC and control fibroblasts (semicircles in A and C ).

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Immuno-Electron Microscopy, Staining, Control, Labeling

Journal: Orphanet Journal of Rare Diseases

Article Title: Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

doi: 10.1186/1750-1172-9-64

Figure Lengend Snippet: Determination of cholesterol content in NPC cells by cellular ELISA. (A) Binding of GST-PFO to cholesterol increases proportionally to the number of cells in the sample within the range of 1-15×10 3 cells/well. Triton X-100-permeabilized NPC fibroblasts were exposed to 5 μg/ml GST-PFO followed by anti-GST IgY-peroxidase. Absorbance of the product of peroxidase activity is shown. (B) Permeabilization of cells with Triton X-100 does not extract cholesterol from deposits. Fixed NPC cells were treated with 0.03%-0.2% Triton-X-100 (TX) or 0.05% digitonin, as indicated, and processed for cellular ELISA using GST-PFO. In a series of experiments, prior to labeling with GST-PFO, cells were incubated with 3 mM methyl-β (CDX) for 60 min at 37°C. In these conditions prominent reduction in the binding of GST-PFO to cells is observed. (C) Significant binding of GST-PFO to NPC fibroblasts. NPC cells and healthy controls were processed for cellular ELISA. When indicated, prior to labeling with GST-PFO cells were incubated with 3 mM methyl-β-cyclodextrin. The results are presented as a ratio of absorbance at 450 nm and 595 nm. Data are means ± SEM from four experiments. (D-D’) In non-permeabilized cells cholesterol deposits are not stained with GST-PFO (D) but are labeled with filipin (D’) . (D”) : merged images of D and D’ . Scale bar, 20 μm.

Article Snippet: In studies on colocalization of PFO-stained structures, the anti-GST antibody was accompanied by: (i) rabbit anti-LAMP-1 IgG (Santa Cruz Biotechnology), (ii) rabbit anti-golgin-84 IgG (Santa Cruz Biotechnology), (iii) rabbit anti-protein disulfide isomerase (PDI; Cell Signaling), (iv) mouse anti-peroxisomal membrane protein 70 (PMP70) (Sigma), (v) mouse anti-LBPA IgG (clone 6C4; Echelon), or (vi) phalloidin-FITC (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Labeling, Incubation, Staining