Journal: International journal of molecular sciences

Article Title: Exploration of Bromodomain Proteins as Drug Targets for Niemann-Pick Type C Disease.

doi: 10.3390/ijms26125769

Figure Lengend Snippet: Figure 2. JQ1 enhances NPC1 protein levels in cultured human skin fibroblasts: (A,B) Levels of NPC1 protein in primary cultures of skin fibroblasts from a healthy donor (H; GM05659; black) and a NPCD patient (P; GM018453; orange) in untreated cells (A) and after treatment with JQ1 or vehicle (DMSO) of patient-derived cells for indicated periods and concentrations (B). Values in (A,B) were normalized to levels of vinculin (VCL) protein and to vehicle-treated (JQ1 concentration zero) control

Article Snippet: Membranes were blocked with fat-free milk (5% in Tris-buffered saline 0.138 M NaCl, 0.027 M KCl, 0.025 M Tris-HCl, and 0.05% Tween-20, pH 6.8) for 1 h at room temperature, exposed to antibodies against NPC1 (1:1000; #NB400148, Novus Biologicals / Bio-Techne S.A.S.

Techniques: Cell Culture, Derivative Assay, Concentration Assay, Control

Journal: International journal of molecular sciences

Article Title: Exploration of Bromodomain Proteins as Drug Targets for Niemann-Pick Type C Disease.

doi: 10.3390/ijms26125769

Figure Lengend Snippet: Figure 3. Effects of JQ1 on subcellular distribution of NPC1 in cultured human skin fibroblasts: (A) Fluorescence micrographs of cultured fibroblasts from a healthy donor (top) (GM056549) and a NPCD patient (GM18453) following treatment for 72 h with vehicle (DMSO) (middle) or with JQ1

Article Snippet: Membranes were blocked with fat-free milk (5% in Tris-buffered saline 0.138 M NaCl, 0.027 M KCl, 0.025 M Tris-HCl, and 0.05% Tween-20, pH 6.8) for 1 h at room temperature, exposed to antibodies against NPC1 (1:1000; #NB400148, Novus Biologicals / Bio-Techne S.A.S.

Techniques: Cell Culture, Fluorescence

Journal: International journal of molecular sciences

Article Title: Exploration of Bromodomain Proteins as Drug Targets for Niemann-Pick Type C Disease.

doi: 10.3390/ijms26125769

Figure Lengend Snippet: Figure 6. JQ1 enhances NPC1 levels and reduces cholesterol accumulation in cultured skin fibroblasts in a patient-specific manner: (A,B) Levels of NPC1 protein in primary cultures of skin fibroblasts from different NPCD patients under basal levels (A) and after treatment with JQ1 or vehicle (DMSO) at indicated concentrations for 72 h (B). Top, representative images of immunoblots showing bands corresponding to NPC1 and vinculin (VCL). Bottom, column plots in (A,B) showing mean values normalized to VCL levels [(A): n = 4 preparations] and to vehicle-treated (JQ1 concentration zero) control cultures [(B): n = 3–5 preparations], respectively. Asterisks in (B) indicate statistically signifi- cant changes compared to vehicle control (*, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way ANOVA with Tukey’s post hoc test). (C) Fluorescence micrographs of cultured fibroblasts from a healthy donor and from different NPCD patients (indicated by codes) showing basal levels of unesterified cholesterol without (left; Con) and with JQ1 treatment (right; JQ1; 3 µM for 168 h). Cells were subjected to chemical fixation and cytochemical staining with filipin. Scale bar: 20 µm. Boxplots showing densities

Article Snippet: Membranes were blocked with fat-free milk (5% in Tris-buffered saline 0.138 M NaCl, 0.027 M KCl, 0.025 M Tris-HCl, and 0.05% Tween-20, pH 6.8) for 1 h at room temperature, exposed to antibodies against NPC1 (1:1000; #NB400148, Novus Biologicals / Bio-Techne S.A.S.

Techniques: Cell Culture, Western Blot, Concentration Assay, Control, Fluorescence, Staining

Journal: International journal of molecular sciences

Article Title: Exploration of Bromodomain Proteins as Drug Targets for Niemann-Pick Type C Disease.

doi: 10.3390/ijms26125769

Figure Lengend Snippet: Figure 7. Effects of JQ1 on cholesterol accumulation in cultured skin fibroblasts in the presence of the NPC1 inhibitor U18 or of an HDAC inhibitor: (A) Fluorescence micrographs of cultured fibroblasts

Article Snippet: Membranes were blocked with fat-free milk (5% in Tris-buffered saline 0.138 M NaCl, 0.027 M KCl, 0.025 M Tris-HCl, and 0.05% Tween-20, pH 6.8) for 1 h at room temperature, exposed to antibodies against NPC1 (1:1000; #NB400148, Novus Biologicals / Bio-Techne S.A.S.

Techniques: Cell Culture, Fluorescence

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 2. Identification of Sterol Derivatives with Greater Potency by Means of Chemical Optimization (A) Structures of representative sterol derivatives used in this article. (B) NPC1I1061T colocalization assay, showing dose-response curves for selected oxysterols and sterol derivatives. The extent of colocalization of the NPC1 mutant and LAMP1 was quantified as described in Experimental Procedures, and the data points represent the averages (n = 10) with SE depicted by error bars. (C) Representative images of the experiments in (B). Calibration bar represents 20 mm. See also Figure S1A.

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Mutagenesis

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 3. Effect of Oxysterol Derivatives on Steady-State Expression Level and Maturation Status of NPC1I1061T Mutant (A) The steady-state expression level of FLAG-NPC1-GFP (WT or I1061T) was quantified by measuring GFP fluorescence in the lysate with or without oxysterol derivatives. The GFP fluorescence was normalized with respect to total protein concentration. Data points represent the averages (n = 3) with SD depicted by error bars. (B) Acquisition of EndoH resistance upon treatment with 25HC and its derivative. Cells stably expressing either WT or I1061T version of FLAG-NPC1-GFP were treated as indicated for 24 hr and lysed. The lysates were digested with EndoH and immunoprecipitated with anti-FLAG beads. The immunoprecipitated proteins were subjected to western blot analysis (immunoblotted with anti-FLAG antibody).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Expressing, Mutagenesis, Protein Concentration, Stable Transfection, Immunoprecipitation, Western Blot

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 5. Functional Rescue of Patient- Derived Fibroblasts (A) Comparison of the expression levels and band patterns of endogenous WT (HEK293) and I1061T NPC1 proteins (NPC fibroblast). The filled arrow- head indicates the mature form, and the open arrowhead indicates the immature form. (B) Effects of 25HC and mo56HC on expression level and band pattern of endogenous NPC1I1061T. NPC fibroblasts were treated with the indicated compound for 48 hr and processed for western blot analysis using anti-NPC1 antibody. (C) Effect of other sterol derivatives on expression level and band pattern of I1061T mutant. To facil- itate comparison between the compounds, the concentrations normalized with their EC50s are also shown. (D) Alleviation of intracellular cholesterol accumu- lation by oxysterol derivative. NPC fibroblasts were cultured in the presence of the indicated compound for 48 hr, and processed for filipin staining. Calibration bar represents 100 mm. The intracellular cholesterol accumulation was quanti- fied as described in Experimental Procedures. Error bar represents SD (n = 12). See also Figure S2.

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Functional Assay, Derivative Assay, Comparison, Expressing, Western Blot, Mutagenesis, Cell Culture, Staining

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 6. Dispensability of NTD for Oxysterol Derivative-Mediated Rescue of Mutant NPC1 Protein (A) Schematic representation of the NTD-deleted NPC1-GFP (DNTD). (B) Subcellular localization of the DNTD-WT and DNTD-I1061T. Cells stably expressing the DNTD-NPC1-GFP construct were treated as indicated for 24 hr and colocalization of the NPC1 with LAMP1 was examined. Calibration bar represents 20 mm. (C) Dose-dependent rescue of DNTD-I1061T localization by representative oxysterol derivatives. The graph shows the dose-response curves of representative compounds and the table shows calculated EC50 values. For 25HC, the extrapolated value is shown. Error bar, SE (n = 12).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Mutagenesis, Stable Transfection, Expressing, Construct

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 7. Existence of Non-NTD Sterol-Binding Site (A) Sterol-mediated stabilization of DNTD-I1061T. The steady-state expression level of DNTD-I1061T was quantified as in Figure 3A. Error bar, SD (n = 3). (B) Schematic representation of the NTD-tail-GFP construct. See also Figure S3. (C) Photoaffinity labeling experiments of NTD-deleted NPC1 and NTD-tail NPC1. Membranes from cells stably expressing either FLAG-tagged DNTD-I1061T or NTD-tail-GFP were labeled with mo56AZK as in Figure 4. Right panel shows the labeling of DNTD-WT. Because of the low expression level of the stable cell line, the longer exposure time was used for DNTD-WT. ns, nonspecific labeling/staining. See also Figure S3C. (D) The quantified results of (C).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Binding Assay, Expressing, Construct, Labeling, Stable Transfection, Staining

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Western blot of wild type (W) littermates, NPC1 (Mut) or NPC2 (Mut) mutant mouse lungs using anti-NPC1 or -NPC2 antibody. β-actin used as a loading control. 30 µg protein/lane.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Western Blot, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Lungs from BALB/c wild type (95 days), NPC1 mutant (70 days) and NPC2 mutant mice (88 days). NPC1 and NPC2 mutant mice show “nests” of macrophages and alveolar macrophages with large inclusions.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Wild type lung (A–C). A. Overview of section of lung with alveolar type II cell (AT2) and endothelial cell (Endo). B. Capillary (Ca). C. Respiratory membrane with type I cell (T1) and endothelial cell (Endo). AS, alveolar space. NPC1 mutant lung (D–H). D. Overview of lung with leukocytes in the capillary and excess surfactant in alveolar space. AL, alveolar lipidosis. d. Enlargement of area in D showing vacuolar leukocyte (L). Similar leukocytes were seen in NPC2 mutant lung. E. Alveolar type II cell (AT2) with a foamy alveolar macrophage (AM) in close proximity in the alveolar space. e. Enlargement of area in E showing type II cell-macrophage contact. *Indicates vacuolar inclusions in endothelial cell. F. Alveolar macrophage with lipid-like material and vacuolar inclusions. G. Type II cells with excess surfactant (white arrowhead). H. Endothelial cell with vesicular inclusions (*). NPC2 mutant lung (I–P). I. Overview of lung with surfactant completely filling the alveolar space characteristic of alveolar lipidosis. J. AT2 with an alveolar macrophage containing multivesicular whirls and a foamy circulating macrophage (CM). K. Respiratory membrane of endothelial cell, basement membrane (BM) and type I cell and demonstrating large amounts of surfactant as tubular myelin (TM) and aggregate (Ag) structures. L. Endothelial cell with vesicular structures. M. Alveolar space with black arrowhead indicating proteinaceous material. Similar material was seen in NPC1 mutant lung. N. Large aggregate structure with tightly packed phospholipid-type whirls (gray arrowheads) or string-like structures (white arrowhead) in alveolar space. O. Surfactant vesicles (white arrowheads) filling the alveolar space. P. Type II cell with inset (p) showing autophagosome-like structures (ap) in enlargement.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Membrane, Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Using electron microscopic photographs of type II cells from wild type, NPC1 and NPC2 mutant mice, the size of the lamellar bodies was analyzed using ImageJ. Top. Electron micrographs of typical type II cells from wild type (WT, left), NPC1 (middle) and NPC2 (right) mutant mice. LB, lamellar body. Bottom. Histogram of the lamellar bodies from wild type (white triangles), NPC1 (gray circles) and NPC2 (black circles) type II cells. Frequency of each lamellar body size in micron 2 is expressed as a % of the total numbers of lamellar bodies. Inset: size of lamellar bodies in grouped bins. Wild type, 60 type II cells, 451 LBs, 3 mice; NPC1, 53 type II cells, 435 LBs, 3 mice; NPC2, 37 type II cells, 459 LBs, 3 mice. *Statistically significant difference, P<0.05. E. Lamellar body sizes in grouped bins of ranges of areas.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A–C Cholesterol and phospholipid content of mice lungs. A. Lipid content of the lavaged lung as µg lipid/mg lung protein. Data are mean±SE, n = 4 separate mice. B. Broncho-alveolar lavage (BAL) of mice lungs. Data as µg lipid/gm weight of lung and are mean±SE, n = 4 separate mice. C. Lipid content of lamellar bodies isolated from the lungs of the wild type and mutant mice. Four (NPC1 or wild type littermates) or three (NPC2 or wild type littermates) lamellar body preparation isolated from the lungs of 2–6 mice. *Statistically significant difference versus wild type littermates, # statistically significant difference between wild types. δ Statistically significant difference between mutants, P <0.05. D. Degradation of 3 H-labeled DPPC as percentage of total label uptake. 3 H-DPPC liposome degradation by the isolated, perfused lungs of three wild type or NPC1 mutant mice after intratracheal instillation. Values are means±SE. *Statistically significant difference from wild type. P<0.001, n = 3. Total degradation is the sum of lysophosphatidyl choline (lysoPC), unsaturated PC (unsatPC), and the aqueous fractions.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Mutagenesis, Labeling

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Physical parameters of NPC mice and wild type littermates.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques:

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Left, Western blots of SP-A or actin. Right, quantitation of Western blots. A. Arbitrary units (AU) of SP-A relative to actin from lungs of NPC1 or NPC2 mutant mice (C1 or C2) or age-matched wild type controls (W1 or W2) (n = 6–8) or B. Arbitrary units of SP-A in surfactant (n = 4–9). All samples were loaded at equal protein values. *Significant difference from wild type, ( P <0.05). The two SP-A bands are due to differences in glycosylation.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Western Blot, Quantitation Assay, Mutagenesis, Glycoproteomics

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A. Type II cells were isolated from NPC1 (Top) or NPC2 (Bottom) mutant mice or their corresponding wild type littermates and placed in culture for 24 hrs. The cells were fixed and stained with anti-ABCA3 antibody (green) to mark the lamellar body limiting membrane or filipin (Fil, gray or blue) to mark cholesterol. The merged pictures with anti-ABCA3 in green and filipin in blue are enlarged. Scale bar = 5 µm. B . Alveolar macrophages from NPC mutant mice contain cholesterol. Alveolar macrophages were isolated from the lung lavage from NPC1 (Top) or NPC2 (Bottom) mutant mice or their corresponding wild type littermates and placed in culture for 2 hrs. The cells were fixed and stained with filipin which labels free unesterified cholesterol. Lt, Phase micrograph; Fil, Filipin stain in gray. Merge of phase and filipin (blue) are enlarged. Scale bar = 10 µm.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Mutagenesis, Staining, Membrane

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Alveolar macrophages were isolated and cultured as in Fig. 7. The cells were harvested and the phospholipid and cholesterol content analyzed. W1,W2. Wild type littermates from NPC1 or NPC2 mutant mice, respectively. C1, NPC1; C2, NPC2. The data are mean±SE, n = 4 separate mice. *Statistically significant difference from corresponding wild type littermates. # Significant difference between NPC1 and NPC2 mutant macrophages, P<0.05.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Cell Culture, Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A. Histology of feline lung. Wild type (WT) and NPC1 mutant (NPC1) feline lungs (25 weeks old) stained with hematoxylin and eosin. NPC1 mutant feline show thickened septae, enlarged “foamy” macrophages in capillaries and alveolar macrophages in the alveolar space. B. Electron micrographs (EM) of typical type II cells from wild type (WT) and NPC1 mutant (NPC1) feline indicating that the size of NPC1 mutant feline lamellar bodies is enlarged. C . Histogram of the lamellar bodies from wild type (white triangles) and NPC1 (gray circles) type II cells. Using electron microscopic photographs of type II cells from wild type and NPC1 mutant felines, the size of the lamellar bodies was analyzed using ImageJ. C , Inset: size of lamellar bodies in grouped bins in ranges of areas. Frequency of each lamellar body size in micron 2 is expressed as a % of the total numbers of lamellar bodies. Wild type (WT): 28 type II cells, 250 LBs, 3 feline; NPC1 mutant: 35 type II cells, 323 LBs, 3 feline.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Mutagenesis, Staining

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A. Lipid content of the lung. Data are mean±SE, n = 3 feline. B. Lipid content of lamellar bodies isolated from the lungs of the wild type and NPC1 mutant feline. Data are the mean±SE and range of 5 (WT) or 4 (NPC1 mut) lamellar body preparations isolated from feline lungs analyzed in triplicate. *Significant different from WT.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Mutagenesis

Journal: Cancers

Article Title: NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer

doi: 10.3390/cancers14143543

Figure Lengend Snippet: NPC1 is significantly elevated in aggressive breast cancers and is directly targeted by miR-200c. ( A ) Effect of miR-200c on NPC1 in three TNBC cell lines by qPCR after a 48-h transfection. Unpaired t -Test ( B ) Effect of miR-200c on NPC1 following a 72-h transfection. ( C ) Immunohistochemistry of NPC1 in BT549 (/+ miR-200c, scale bar = 50 µm. ( D ) Luciferase assay performed on wildtype (WT) or mutated (Mut) NPC1 3’UTR, with and without exogenous miR-200c. (Student’s t -Test) ( E ) Western blot of NPC1 in serum-free media (SFM) or lipoprotein depleted serum (LPDS) for 24 h, in two TNBC cell lines. ( F ) Effect of miR-200c on NPC1 induction by a 24-h SFM and LPDS treatment. miR-200c transfection = 96 h. ( G ) NPC1 in 2019 METABRIC breast cancer cohort by subtype (left, One-way ANOVA with Tukey Test, relative to “normal”) and grade (right, one-way ANOVA with Tukey Test). ( H ) Western blot of baseline NPC1, a panel of breast cancer cell lines. p -values denoted by asterisks where * ≤ 0.05; ** ≤ 0.01; **** ≤ 0.0001; or ns = not significant (>0.05).

Article Snippet: The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868).

Techniques: Transfection, Immunohistochemistry, Luciferase, Western Blot

Journal: Cancers

Article Title: NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer

doi: 10.3390/cancers14143543

Figure Lengend Snippet: NPC1 supports breast cancer cell invasion and growth in soft agar. ( A ) Western blot confirmation of NPC1 knockdown by shRNA. NPC1 normalized to GAPDH. ( B ) Relative confluency of three TNBC cell lines with two unique shRNAs, following 72 h in 2D cultured, as measured by xCELLigence. ( C ) Growth of cell lines over 72 h, as measured by xCELLigence. ( D ) Sum159PT colony formation in soft agar, two weeks after seeding 3,000 cells/well. ( E ) Invasion of TNBC cells through Cultrex after 24 h, from serum-free DMEM toward 10% DMEM. ( F ) MCF7 colony formation in soft agar, two weeks after seeding 35,000 cells/well. ( G ) MCF7 invasion through Cultrex gel over 24 h. MCF7 cells transfected with an empty vector (pcDNA3.1) or constitutively active NPC1 (pcDNA3.1-NPC1). All statistics: student’s unpaired t -Test. p -values denoted by asterisks where * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001; **** ≤ 0.0001.

Article Snippet: The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868).

Techniques: Western Blot, shRNA, Cell Culture, Transfection, Plasmid Preparation

Journal: Cancers

Article Title: NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer

doi: 10.3390/cancers14143543

Figure Lengend Snippet: TNBC cells are cholesterol auxotrophs, but do not require NPC1 for adequate cholesterol supply. ( A ) Growth of breast cancer cell lines over 7 days in cholesterol-deplete (5% LPDS) relative to cholesterol-replete (5% LPDS + 10 µg/mL LDL) media. ( B ) Colony formation in soft agar, cholesterol-deplete ( top ) and –replete ( bottom , 10 µg/mL LDL); fourteen days. ( C ) Basal levels of key cholesterol metabolism proteins NPC1, LDLR, and HMGCR in a panel of breast cancer cells. ( D ) Correlation of NPC1 protein levels (quantified from western, 3C) to cell viability in LPDS (viability score from 3A). ( E ) Western blot of key cholesterol proteins in shRNA#1 cell lines. ( F ) Uptake of fluorescent dil-LDL following 6 h of incubation, analyzed by a plate reader and normalized to protein content. ( G ) Growth of Sum159PT cells in soft agar when supplemented with MBCD:Cholesterol (1% MβCD complexed with 10 µg/mL cholesterol) over 14 days (Two-way ANOVA, Tukey Test). p -values denoted by asterisks where ** ≤ 0.01; or ns = not significant (>0.05).

Article Snippet: The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868).

Techniques: Western Blot, shRNA, Incubation

Journal: Cancers

Article Title: NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer

doi: 10.3390/cancers14143543

Figure Lengend Snippet: NPC1 supports mitochondrial respiration and maintains mitochondrial morphology. ( A ) Oxygen consumption rate (OCR) during Seahorse mitochondrial stress test, performed on Sum159PT and MDA-231 cell lines with NPC1 knockdown. ( B ) Mitochondrial stress test performed on MCF7 with pcDNA3.1-NPC1 or an empty vector. ( C ) Quantification of basal OCR and spare respiratory capacity of TNBC cells from mitochondrial stress test shown in A. ( D ) Mitochondrial ROS was evaluated using mitoSox staining. ( E ) Top: Representative images of fluorescence microscopy of TNBC cells stained with MTC02 (mitochondrial antibody). Bottom: quantification of cells with mitochondrial morphology defined as either elongated, intermediate, or short. ( F ) Confocal Z-stacking of mitochondria stained with MTC02, with representative images of ”elongated” (shCTRL) versus “shortened” (shNPC1) mitochondria. A snapshot of 60× with 4× zoom (top panel), and 60× with 8× zoom (bottom panel). ( G ) Quantification of mitochondrial elongation, as quantified by ImageJ. ( H ) Representative transmission electron microscopy image of short (“S”) and donut (“D”) mitochondria in Sum159PT with shNPC1 (30,000×). p -values denoted by asterisks where * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001.

Article Snippet: The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868).

Techniques: Plasmid Preparation, Staining, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy

Journal: Cancers

Article Title: NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer

doi: 10.3390/cancers14143543

Figure Lengend Snippet: NPC1 affects mTOR and autophagy signaling under stress conditions in TNBC. ( A ) Western pospho-S6K (T389) signaling under cholesterol-stressed conditions. Control cells (lane 1, 4) were maintained in full serum. Where indicated, MβCD is used to deplete cholesterol for 2 h and then, as indicated (lane 3, 6), MβCD is removed and replaced with MβCD:Cholesterol to replenish cholesterol supply. Numbers below pS6K1 represent levels normalized to total S6K1. ( B ) S6 kinase signaling under metabolic or lysosome stress conditions for 24 h (LPDS−5% lipoprotein depleted serum; AAS-amino acid depletion with 5% FBS; BafA1-5nM). pS6K (Ser235/236) and pS6 are normalized to their total controls. NPC1 is normalized to GAPDH. ( C ) Autophagy markers p62 and LC3B under metabolic or lysosomal stress conditions.

Article Snippet: The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868).

Techniques: Western Blot

Journal: Cancers

Article Title: NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer

doi: 10.3390/cancers14143543

Figure Lengend Snippet: NPC1 has therapeutic potential alone and in combination with chemotherapies in TNBC. ( A ) A graphical summary of the anti-tumor effects of NPC1 silencing. Created with biorender.com ( B ) Dose-response curves of U18666A in TNBC (MDA-MB-231, BT549) and ER+ (T47D), 48 h drug treatment as analyzed by crystal violet. Linear regression with a variable slope. ( C ) Additive and synergistic effects of U18666A and Paclitaxel in BT549 cells. Three doses of each drug were given for 48 h and analyzed by crystal violet. The additive/synergy effect was analyzed and calculated using SynergyFinder 2.0.

Article Snippet: The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868).

Techniques:

Journal: Life Science Alliance

Article Title: Unbiased yeast screens identify cellular pathways affected in Niemann–Pick disease type C

doi: 10.26508/lsa.201800253

Figure Lengend Snippet: (A) Ncr1 was tagged with one half of the DHFR enzyme and mated to strains carrying fusion proteins to the other half of the enzyme. Interaction strength was a function of colony size of the diploids on methotrexate divided by their abundance. Ncr1 had high interaction strength with Pmc1 and Fth1 relative to the panel of vacuolar proteins. (B) Pmc1 was tagged with one half of the DHFR enzyme and mated to strains carrying fusion proteins to the other half of the enzyme and had high interaction strengths with Fth11, Apc11, and Ncr1 relative to the panel of vacuolar proteins. Means ± SD, N = 8. (C) Graphic of predicted complex of Ncr1, Pmc1, Apc11, and Fth1. (D) Co-immunoprecipitation of NPC1 and PMCA2 (ATP2B2) from rat cerebellum. (E) qRT-PCR for mRNA expression of PMCA2 (ATP2B2) in wild-type, heterozygous, and homozygous cerebellums of 8-wk Npc1 nih balb/c mice. N = 5, *** P = 0.0004 as compared with WT, calculated by one-way ANOVA.

Article Snippet: 500 μl of each supernatant was incubated, overnight at 4°C, with SureBeads Protein A magnetic beads (Bio-Rad) previously bound to 10 μg of NPC1 (NB400-148; Novus Bioscience) rabbit polyclonal antibody or 10 μg of IgG from rabbit serum (Sigma-Aldrich), following the manufacturer’s instructions.

Techniques: Immunoprecipitation, Quantitative RT-PCR, Expressing

Journal: Life Science Alliance

Article Title: Unbiased yeast screens identify cellular pathways affected in Niemann–Pick disease type C

doi: 10.26508/lsa.201800253

Figure Lengend Snippet: (A) Serial dilution of yeast strains was performed on ∆ ncr1 background with either MAC1 or FZO on GAL1pr repressible promoter and grown in glucose to inhibit protein expression. (B) The relative mitochondrial DNA (mtDNA) content, TMRM intensity, and perinuclear distribution of mitochondria were measured in control and NPC patient cells. Mean ± SD. N = 100 ** P < 0.01, *** P < 0.001, **** P < 0.0001; t test. (C) Representative EM images of control (a) and NPC (b) mitochondria. Scale bar = 1 μm. (D) Statistical distribution of the length in sections of control NPC1 +/+ (open columns) and NPC 1 −/− patient (filled columns) mitochondria acquired from analyses of EM data. **** P < 0.0001, calculated by unpaired t test with Welch’s correction. (E) Serial dilution of ∆ ncr1 on GAL1pr-FZO, FIS1 background in glucose to inhibit protein expression.

Article Snippet: 500 μl of each supernatant was incubated, overnight at 4°C, with SureBeads Protein A magnetic beads (Bio-Rad) previously bound to 10 μg of NPC1 (NB400-148; Novus Bioscience) rabbit polyclonal antibody or 10 μg of IgG from rabbit serum (Sigma-Aldrich), following the manufacturer’s instructions.

Techniques: Serial Dilution, Expressing

Journal: Life Science Alliance

Article Title: Unbiased yeast screens identify cellular pathways affected in Niemann–Pick disease type C

doi: 10.26508/lsa.201800253

Figure Lengend Snippet: ∆ncr1 yeast do not exhibit gross mitochondrial defects, despite oxygen consumpt ion being altered in NPC1 CHO cells. (A) Control (BY4741) and ∆ ncr1 colonies were grown on SD complete (in mid logarithmic growth or during stationary) or on S-Gal complete, and no gross mitochondrial defects were observed. Scale bar: 10 μM. (B) ∆ ncr1 colonies were grown on a non-fermentable source (glycerol), and no growth defects where observed as compared with BY4741. ∆ mmm1 colonies, which cannot grow on glycerol, are used as a positive control. (C) Oxygen consumption in wild-type and mutant NPC1 CHO cells cultured in reduced glucose was determined using MitoXpress-Xtra assay. Mean ± SD. N = 100 *** P < 0.001; t test.

Article Snippet: 500 μl of each supernatant was incubated, overnight at 4°C, with SureBeads Protein A magnetic beads (Bio-Rad) previously bound to 10 μg of NPC1 (NB400-148; Novus Bioscience) rabbit polyclonal antibody or 10 μg of IgG from rabbit serum (Sigma-Aldrich), following the manufacturer’s instructions.

Techniques: Positive Control, Mutagenesis, Cell Culture

Journal: Life Science Alliance

Article Title: Unbiased yeast screens identify cellular pathways affected in Niemann–Pick disease type C

doi: 10.26508/lsa.201800253

Figure Lengend Snippet: (A) Representative images from GFP localization screen including Prk1-GFP and She4-GFP in control (BY4741) relative to ∆ ncr1 yeast. Prk1 is localized to the actin cortical patch in BY4741 yeast, but in the ∆ ncr1 yeast Prk1 is found to be diffuse and mainly cytoplasmic. She4 is localized primarily to the cytoplasm where in the ∆ ncr1 yeast, the localization is more punctate. (B) Representative images of NPC1 and control patient fibroblasts stained for acetylated α-tubulin. Scale bar: 10 μm. (C) Graphical representation of the fluorescent intensities of acetylated α-tubulin. Mean ± SD. N = 10–20 ** P < 0.01; t test. (D) Protein levels of α-tubulin, acetylated α-tubulin, αTAT1, HDAC6, and SIRT2 were measured in control and NPC1 patient fibroblasts. (D, E) Quantitation of blots from (D) by densitometry. (F) Treatment with 125 μM NAD for 24 h reduced acetylated α-tubulin expression in NPC1 patient cells, fluorescence measured using a flow cytometer and analysed with FloJo software. Mean ± SD, N = 3 * P < 0.05; one-way ANOVA.

Article Snippet: 500 μl of each supernatant was incubated, overnight at 4°C, with SureBeads Protein A magnetic beads (Bio-Rad) previously bound to 10 μg of NPC1 (NB400-148; Novus Bioscience) rabbit polyclonal antibody or 10 μg of IgG from rabbit serum (Sigma-Aldrich), following the manufacturer’s instructions.

Techniques: Staining, Quantitation Assay, Expressing, Fluorescence, Flow Cytometry, Software

Journal: Life Science Alliance

Article Title: Unbiased yeast screens identify cellular pathways affected in Niemann–Pick disease type C

doi: 10.26508/lsa.201800253

Figure Lengend Snippet: (A) Representative images of control and NPC1 patient fibroblasts treated with 50 μM miglustat or 250 μM HPβCD stained for acetylated α-tubulin. Scale bar: 10 μm. (B) Quantification of the fluorescent intensities of acetylated α-tubulin. Mean ± SD. N = 10–20 **** P < 0.0001, ns P > 0.05 two-way ANOVA.

Article Snippet: 500 μl of each supernatant was incubated, overnight at 4°C, with SureBeads Protein A magnetic beads (Bio-Rad) previously bound to 10 μg of NPC1 (NB400-148; Novus Bioscience) rabbit polyclonal antibody or 10 μg of IgG from rabbit serum (Sigma-Aldrich), following the manufacturer’s instructions.

Techniques: Staining