Journal: Journal of Cell Communication and Signaling

Article Title: LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells

doi: 10.1007/s12079-020-00551-w

Figure Lengend Snippet: Lipid rafts localization of LRP6 in SK-N-BE2 cells. a-e SK-N-BE2 cells, either untreated or treated with 10 nM tPA for 10 min at 37 °C, were lysed and the supernatant fraction was subjected to sucrose density gradient. After centrifugation, the gradient was fractionated and each fraction was recovered and analyzed by western blot using anti-LRP6 pAb (a), anti-phospho LRP6 pAb (p-LRP6) (b), anti-phospho β-catenin (p-β-catenin) pAb (c), anti-CD71 mAb (d) or an anti-flotillin pAb (e). Right panel: Densitometric analysis of sucrose gradient fractions. The columns indicate the percent distribution across the gel of raft-fractions 4–5 and 6 (Triton X-100-insoluble fractions) and 9–10 and 11 (Triton X-100-soluble fractions), as detected by densitometric scanning analysis. Results represent the mean ± SD from three independent experiments (*) p < 0.01 TX-100-insoluble fractions from tPA-treated cells vs TX-100-insoluble from control cells; (°) p < 0.01 TX-100-soluble fractions from tPA-treated cells vs TX-100-soluble from control cells. f SK-N-BE2 cells, untreated or treated with 10 nM tPA, were lysed in lysis buffer, followed by immunoprecipitation with goat anti-LRP6 pAb. An IgG isotypic control was employed. The immunoprecipitates were spotted onto nitrocellulose strips and incubated with Cholera Toxin B Subunit-Peroxidase (from Vibrio Cholerae), as described in Materials and Methods. As a control, the immunoprecipitates were assessed by Dot-blot with anti-LRP6 mAb. A representative experiment among three is shown. Bar graph in the right panel shows densitometric analysis. Results represent the mean ± SD from three independent experiments. (*) p < 0.01 tPA vs control cells

Article Snippet: The beads were removed by centrifugation (500×g for 1 min) and the supernatants were incubated with goat anti-LRP6 Ab (R&D Systems, Minneapolis, MI, USA) at 4 °C overnight.

Techniques: Centrifugation, Western Blot, Control, Lysis, Immunoprecipitation, Incubation, Dot Blot

Journal: Journal of Cell Communication and Signaling

Article Title: LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells

doi: 10.1007/s12079-020-00551-w

Figure Lengend Snippet: Effect of LRP1 silencing on LRP6 phosphorylation induced by tPA in SK- N BE2 cells. a SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with siRNA LRP1, were analyzed by Western blot, using anti-phospho LRP6 pAb. PVDF was stripped and analyzed with anti-total LRP6 pAb. Densitometric analysis is shown. Results represent the mean ± SD from 3 independent experiments. (*) p < 0.01 scrambled SiRNA +tPA treated cells vs scrambled SiRNA, (°) p < 0.01 SiRNA LRP1+ tPA treated cells vs SiRNA LRP1. b Evaluation of LRP1 expression after 72 h siRNA transfection by Western blot analysis using anti-LRP1 pAb. PVDF was stripped and analyzed with anti-actin mAb. A scrambled siRNA was used as control. Bar graph to the right shows densitometric analysis. Results represent the mean ± SD from three independent experiments. (*) p < 0.01 SiRNA LRP1 vs scrambled siRNA

Article Snippet: The beads were removed by centrifugation (500×g for 1 min) and the supernatants were incubated with goat anti-LRP6 Ab (R&D Systems, Minneapolis, MI, USA) at 4 °C overnight.

Techniques: Phospho-proteomics, Western Blot, Expressing, Transfection, Control

Journal: Journal of Cell Communication and Signaling

Article Title: LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells

doi: 10.1007/s12079-020-00551-w

Figure Lengend Snippet: Involvement of “lipid rafts” on LRP6 phosphorylation and β-catenin phosphorylation induced by tPA in SK-N BE2 cells. a SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with 5 mM MβCD, were analyzed by Western blot, using anti-phospho LRP-6 pAb. PVDF was stripped and analyzed with anti-total LRP6 pAb. Densitometric analysis is shown. Results represent the mean ± SD from 3 independent experiments. (*) p < 0.01 tPA treated cells vs untreated cells; (§) p < 0.01 MβCD +tPA treated cells vs tPA treated cells. b SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with 5 mM MβCD were analyzed by Western blot, using anti-phospho β-catenin pAb PVDF was stripped and analyzed with anti-β-catenin mAb. Densitometric analysis is shown. Results represent the mean ± SD from 3 independent experiments. (*) p < 0.01 tPA treated cells vs untreated cells; (§) p < 0.01 MβCD +tPA treated cells vs tPA treated cells; c SK-N-BE2 cells, untreated or treated with 10 nM tPA, in the presence or in the absence of pre-treatment with 5 mM MβCD were lysed in lysis buffer and subjected to cholesterol (CHOL) analysis. Neutral lipid extracts were separated by high-performance thin layer chromatography (HPTLC) using a solvent system of hexane/diethyl ether/acetic acid (70: 30: 1, v/v/v) and detected by staining with 2% copper acetate solution in 8% phosphoric acid and subsequent heating at 120 °C for 15 min. (*) p < 0.01 MβCD treated cells vs untreated cells; (°) p < 0.01 MβCD +tPA treated cells vs tPA treated cells

Article Snippet: The beads were removed by centrifugation (500×g for 1 min) and the supernatants were incubated with goat anti-LRP6 Ab (R&D Systems, Minneapolis, MI, USA) at 4 °C overnight.

Techniques: Phospho-proteomics, Western Blot, Lysis, High Performance Thin Layer Chromatography, Solvent, Staining

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: Pedigree of the family HC438 with the segregation of p.(Val1382Phe) variant in LRP6 , p.(Pro398Ala) variant in CYP7A1 and p.(Ser202His) variant in LDLRAP1 and the weighted Polygenic Risk Score (wPRS). The proband II-7 is indicated by the black arrow. Squares and circles represent men and women, respectively. Affected family members are indicated by values highlighted in gray. More severely affected family members are indicated by bold values. * Age at lipid measurement in years. ** Myocardial infarction at 75 years old. *** Severe atheroma. # Under ciprofibrate. ## Under 5 mg rosuvastatin treatment. Patients for whom ♦ whole exome, □ whole genome sequencing was performed. Lipid values in mmol/L: TC for total cholesterol; LDL-C for LDL cholesterol; HDL-C for HDL cholesterol; TGs for triglycerides.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Variant Assay, Sequencing

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: Variants in CYP7A1 , LDLRAP1 and LRP6 . The pathogenicity of the variants was evaluated using Varsome, PolyPhen2, Provean, ClinVar, CADD score and Splice AI.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques:

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: Biological and clinical characteristics of the affected carriers of CYP7A1, LDLRAP1 and LRP6 variant.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Variant Assay

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: Structure of the LRP6 receptor and position of the identified variants . The LRP6 receptor contains the following structural motifs: signal peptide (SP), 4 β-propeller domains, 4 EGF-like domains (involved in the pH-dependent release of ligands in endosome), 3 LDLR type A repeats (responsible for the binding of ligands), a transmembrane anchor (binds the receptor to the cell membrane), and a cytoplasmic domain with PPPSP motifs (2 motifs at position 1487 and 1604 that allow the receptor to function in the Wnt/β-catenin pathway). Red arrows indicate the position of the variants identified in this study. Figure built from data from UniProt ( www.uniprot.org (accessed on 12 October 2020)) and Ensembl ( www.ensembl.org/index.html (accessed on 12 October 2020)) databases.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Binding Assay, Membrane

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: Crystal structure of wild-type and mutant LRP6-E3E4 with β-propeller domains (green) and epidermal growth factor (EGF)-like domains (gray) . ( A , B ) LRP6-E3E4 Tyr972 residue (red) has polar contacts (yellow dotted lines) with Asp971 and Glu993 (blue). ( C , D ) LRP6-E3E4 mutant Cys972 residue (red) has a polar contact (yellow dotted line) only with Asp971 (blue).

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Mutagenesis, Residue

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: Effect of inhibited, overexpressed or mutated LRP6 in HEK293T and HuH7 cells . ( A ) LRP6 mRNA expression in HuH7 after the silencing of LRP6 . Reactions were run in triplicate for each cDNA. POLR2A was used as the reference housekeeping gene. The relative quantification of gene expression was performed using the ∆∆C T method and non-transfected cells were used for calibration. ( B ) LDL-Bodipy uptake in HuH7 after silencing of LRP6 . Median fluorescence intensity of 50,000 events was acquired for each sample, but only the median fluorescence intensity of living cells is presented. Data represent three independent assays performed in triplicate. ( C ) Expression of WT or mutated LRP6 at the cell surface of transfected HEK293T . The median fluorescence intensity of 100,000 events was acquired for each sample, but only the median fluorescence intensity of living cells is presented. Data represent four independently performed assays. ( D , E ) LRP6 expression in HEK293T cells after transfection with LRP6-WT or mutated plasmid (p.(Thr1479Ile) and p.(Tyr972Cys) variants) . Proteins were extracted from transfected cells, separated by electrophoresis and then transferred onto PVDF membrane. The membrane was incubated with primary antibody (anti-LRP6), followed by incubation with secondary antibody before detection using the iBright TM FL1500 imaging system. Protein was quantified by ImageJ software. Equal loading was confirmed using the ß-actin antibody. Data represent three independent assays. ( F ) LDL uptake in HEK293T after transfection with an empty vector, LRP6-WT or mutated plasmid . The median fluorescence intensity of 100,000 events was acquired for each sample, but only the median fluorescence intensity of living cells is presented. The fluorescence of each sample was normalized using the empty vector (PcM) as a reference. Data represent three independent assays, each performed in triplicate. In all experiments, the difference between conditions was determined by Bonferroni’s Multiple Comparison Test in one-way ANOVA and * p < 0.05, ** p < 0.01, *** p < 0.001 were considered as statistically significant. Results are shown as mean ± SD. Error bars represent ± SD.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Expressing, Quantitative Proteomics, Gene Expression, Transfection, Fluorescence, Plasmid Preparation, Electrophoresis, Membrane, Incubation, Imaging, Software, Comparison

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: LDL receptor expression, LDL binding and uptake, and LRP6 gene expression in patients EBV-transformed B-lymphocytes. ( A ) LDL receptor, ( B ) LDL-Bodipy binding and ( C ) LDL-Bodipy uptake quantification in EBV-transformed B-lymphocytes from normocholesterolemic subjects (N), LDLR mutation carriers (FH), hypercholesterolemic patients without an identified mutation (FH/M-), and two LRP6 -p.(Val1382Phe) carriers from the HC438 family: II-4 and III-6 (see ). The median fluorescence of living cells is presented. Data represent five independently performed assays. ( D ) LRP6 gene expression. Relative Quantification (RQ) of LRP6 in EBV-transformed B-lymphocytes. Reactions were run in triplicate for each cDNA. HPRT and POL2RA were used as reference genes. The relative quantification was performed using the ∆C T method. ( A – D ). Bonferroni’s Multiple Comparison Test in one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Expressing, Binding Assay, Gene Expression, Transformation Assay, Mutagenesis, Fluorescence, Quantitative Proteomics, Comparison

Journal: Metabolites

Article Title: Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia

doi: 10.3390/metabo12030262

Figure Lengend Snippet: LDL-C levels and weighted Polygenic Risk Score (wPRS) comparison among the carriers of CYP7A1 , LRP6 and/or LDLRAP1 variants.

Article Snippet: A human LRP6 APC-conjugated antibody was purchased from the R&D System and its specificity was confirmed in vitro by ELISA.

Techniques: Comparison, Variant Assay

Journal: eLife

Article Title: Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice

doi: 10.7554/elife.46134

Figure Lengend Snippet: Figure 1. Design and validation of FLAgs as activators of the Wnt-bcatenin pathway. (A) Surface plasmon resonance (SPR) binding kinetics of FP Fab. Kinetics were derived from curves for soluble FP Fab interacting with immobilized FZD CRD, and ‘x’ indicates no detectable binding. (B) Anti-LRP6 Ab inhibitory activity. Inhibition of WNT1 or WNT3A signaling by indicated LRP6 Abs in the diabody-Fc format was assessed using stimulation with purified WNT3A or upon WNT1 cDNA transfection. (C) Molecular architecture of tetravalent FLAgs. (D) Activation of bcatenin signaling by FLAgs. Dose response curves are shown for the activation of a LEF/TCF reporter gene (y-axis) in HEK293T cells by serial dilutions of pan-specific FLAg proteins (FP+P- L61+1, FP+P-L63+3 and FP+P-L61+3) (x-axis). Error bars indicate SEM, n = 3. (E) Levels of bcatenin protein in RKO cells after 30 min treatment with the indicated concentrations of pan-FLAg (FP+P-L61+3). Representative blot of three replicates. (F) Time course of bcatenin and phosphorylated Disheveled- 2 (p-DVL2) protein levels in RKO cells treated with 10 nM pan-FLAg (FP+P-L61+3). Representative blot of three replicates. DOI: https://doi.org/10.7554/eLife.46134.002 The following source data and figure supplement are available for figure 1:

Article Snippet: Fc-tagged ECD fusion proteins of human (1505-LR-025) and mouse (2960-LR-025) LRP6 and mouse LRP5 (7344-LR-025/CF) were purchased from R and D Systems.

Techniques: Biomarker Discovery, SPR Assay, Binding Assay, Derivative Assay, Activity Assay, Inhibition, Purification, Transfection, Activation Assay