Journal: Free radical biology & medicine

Article Title: Discovery of sterically-hindered phenol compounds with potent cytoprotective activities against ox-LDL-induced retinal pigment epithelial cell death as a potential pharmacotherapy.

doi: 10.1016/j.freeradbiomed.2021.11.026

Figure Lengend Snippet: Fig. 6. Hindered phenol compound increase NPC1 levels and reduce intracellular ox-LDL caused cholesterol accumulation. (A) Serum starved ARPE-19 cells were treated with indicated doses of PMC for 18 h, cell lysates were collected and NPC1 protein levels were examined by western blot and imaged Li-COR® infrared imaging system. The fluorescent intensity of NPC1 levels were normalized to the fluorescent intensity of α-tubulin to determine fold change. (B) ARPE-19 cells were treated with ox-LDL in the presence or absence of PMC, cell lysates were collected 18 h post treatment and western blot was performed to determine the relative protein levels of NPC-1 using Li- COR® infrared imaging system. The fluorescent intensity of NPC1 levels were normalized to the fluorescent intensity of α-tubulin to determine the relative expression levels of NPC1. (C) ARPE-19 cells were treated with ox-LDL for 8hr, cells were washed and then incubated with or without PMC. Cell lysates were collected and the total cellular cholesterol content was determined by normalizing to total cellular protein content. Serum starved untreated cells served as control. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies: PPARγ and vinculin (catalog # 2443S and 13901S, Cell Signaling, Danvers, MA), NPC1 (Catalog # MAB10105, R&D systems, Inc. Minneapolis, MN), alphatubulin (catalog # CP06, Calbiochem, MilliporeSigma, Burlington, G. Gnanaguru et al. Free Radical Biology and Medicine 178 (2022) 360–368 MA).

Techniques: Western Blot, Imaging, Expressing, Incubation, Control

Journal: Bioorganic chemistry

Article Title: Bioinformatic and biochemical findings disclosed anti-hepatic steatosis mechanism of calycosin.

doi: 10.1016/j.bioorg.2020.103914

Figure Lengend Snippet: Fig. 2. All targets/genes of calycosin and fatty liver were picked up for collection of central biotargets of calycosin for anti-fatty liver, covering ALDH2, NPC1, HMGB1, UGT1A1, MAPK3, EGFR, HTR2, AMIF, CYP19A1.

Article Snippet: The fatty liver sections from biopsy were dewaxed and then blocked with 5% bovine serum albumin solution (Beyotime Biotechnology, China) for about 1 h. After rinse with phosphate buffer saline/0.5% tween 20 solution for about 3 times, the sections were incubated with primary antibodies of ALDH2 (1:200, BOSTER Biological Technology, China), NPC1 (1:200, BOSTER), HMGB1 (1:200, BOSTER) at 4 °C overnight, then the sections being incubated with diluted secondary antibodies of DyLight 488 (1:400, BOSTER Biological Technology, China).

Techniques:

Journal: Bioorganic chemistry

Article Title: Bioinformatic and biochemical findings disclosed anti-hepatic steatosis mechanism of calycosin.

doi: 10.1016/j.bioorg.2020.103914

Figure Lengend Snippet: Fig. 5. Clinical findings of patients with fatty liver. In medical detection, the steatohepatitis patients were identified through B-ultrasound and pathological stain. And the fatty liver sections showed reduced ALDH2, NPC1 expressions and elevated HMGB1expression.

Article Snippet: The fatty liver sections from biopsy were dewaxed and then blocked with 5% bovine serum albumin solution (Beyotime Biotechnology, China) for about 1 h. After rinse with phosphate buffer saline/0.5% tween 20 solution for about 3 times, the sections were incubated with primary antibodies of ALDH2 (1:200, BOSTER Biological Technology, China), NPC1 (1:200, BOSTER), HMGB1 (1:200, BOSTER) at 4 °C overnight, then the sections being incubated with diluted secondary antibodies of DyLight 488 (1:400, BOSTER Biological Technology, China).

Techniques: 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 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: Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques

Article Title: Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

doi: 10.18433/j3m029

Figure Lengend Snippet: Figure 1. Changes in relative NPC1L1, ABCG5, and ABCG8 mRNA levels in the duodenum, jejunum, and ileum of mice 24 h after oral PR administration (5 mg/kg and 15 mg/kg). Data are expressed as means ± standard deviation (SD; n = 4). Significant differences between control and PR-treated mice are shown (*p < 0.05 and **p < 0.01).

Article Snippet: Immunoreactive NPC1L1 proteins were detected using polyclonal NPC1L1 antibodies (NB400-128, Novus Biologicals, Littleton, CO, USA), monoclonal -actin antibodies (Acris Antibodies, Herford, Germany), and an ECL Prime Western Blotting Detection system (GE Healthcare).

Techniques: Standard Deviation, Control

Journal: Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques

Article Title: Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

doi: 10.18433/j3m029

Figure Lengend Snippet: Figure 2. Changes in relative NPC1L1, ABCG5, and ABCG8 mRNA levels in the duodenum of mice 6 h after oral PR administration (5 mg/kg and 15 mg/kg) or 24 h after oral EZ administration (5 mg/kg). Results are expressed as means ± SD (n = 4).

Article Snippet: Immunoreactive NPC1L1 proteins were detected using polyclonal NPC1L1 antibodies (NB400-128, Novus Biologicals, Littleton, CO, USA), monoclonal -actin antibodies (Acris Antibodies, Herford, Germany), and an ECL Prime Western Blotting Detection system (GE Healthcare).

Techniques:

Journal: Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques

Article Title: Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

doi: 10.18433/j3m029

Figure Lengend Snippet: Figure 3 The effects of PR on NPC1L1, ABCG5, and ABCG8 expression in HepG2 cells. (a) Relative NPC1L1, ABCG5, and ABCG8 mRNA levels 24 h after PR (3, 10, 30, and 300 M) treatment. (b) Time course of relative NPC1L1 mRNA expression changes after PR (30 M) treatment. (c) NPC1L1 protein expression after PR (3, 10, 30, and 300 M) treatment. Results are expressed as means ± SD (n = 4). Significant differences between (a) 0 h (control) and each time, (b), and (c) no treatment (control) and PR-treated HepG2 cells are shown (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: Immunoreactive NPC1L1 proteins were detected using polyclonal NPC1L1 antibodies (NB400-128, Novus Biologicals, Littleton, CO, USA), monoclonal -actin antibodies (Acris Antibodies, Herford, Germany), and an ECL Prime Western Blotting Detection system (GE Healthcare).

Techniques: Expressing, Control

Journal: Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques

Article Title: Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

doi: 10.18433/j3m029

Figure Lengend Snippet: Figure 4 Effects of EZ on relative NPC1L1, ABCG5, and ABCG8 mRNA expression 24 h after EZ (3, 10, 30, and 300 M) treatment in HepG2 cells. Results are expressed as means ± SD (n = 4).

Article Snippet: Immunoreactive NPC1L1 proteins were detected using polyclonal NPC1L1 antibodies (NB400-128, Novus Biologicals, Littleton, CO, USA), monoclonal -actin antibodies (Acris Antibodies, Herford, Germany), and an ECL Prime Western Blotting Detection system (GE Healthcare).

Techniques: Expressing

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