Journal: Journal of Biological Chemistry

Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function

doi: 10.1074/jbc.m110.190330

Figure Lengend Snippet: FIGURE 3. LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 m. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transducedwithlentiviralparticlesharboringshRNAagainstLrp4.ReductionofLrp4RNAlevelswasassessedbyreal-timequantitativePCR,priortotheaddition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

Article Snippet: Recombinant human DKK1 and LRP6 were purchased from R&D Systems.

Techniques: Inhibition, In Vitro, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Activity Assay

Journal: Nature communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin.

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: Fig. 6 | Oncogenic APC alters interactions between Wnt receptors and their effectors. To examine the effect of oncogenic APC on interactions between Wnt receptors and key lipids, cells co-expressing EGFP-tagged A, C Fzd7 or B, D LRP6 and mCherry-tagged A, B D4H or C, D pleckstrin homology (PH) domain of phos- pholipase C δ1 (PLC-δ1, PI(4,5)P2 sensor) were used to perform FLIM FRET. E To examine the effect of oncogenic APC on PI(4,5)P2 plasma membrane levels, cells expressing EGFP-tagged PLC-δ1-PH were used to measure membrane-associated EGFP fluorescence intensity using flow cytometry. EGFP plasma membrane fluor- escence intensity was normalized to total EGFP fluorescence intensity. To examine the effect of oncogenic APC on the interactions between Dvl1 and key lipids, cell co- expressing EGFP-tagged F, G Dvl1 and mCherry-tagged F D4H or G PLC-δ1 were used to perform FLIM FRET. YAMC, IMCE,and IMCE βcat cellswere pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or phenylarsine oxide (PAO) (20

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R andDSystems,Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: Expressing, Clinical Proteomics, Membrane, Cytometry

Journal: Nature communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin.

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: Fig. 7 | Oncogenic APC enhances macromolecular interactions within Wnt receptor nanoscale signaling platforms. For in vitro FLIM-FRET experiments, cells co-expressing EGFP- and mCherry-tagged A Fzd7 or B LRP6 or C EGFP-tagged LRP6 and mCherry-tagged Fzd7 were used to perform homo- and hetero-clustering FLIM-FRET analyses, respectively. To examine the effect of oncogenic APC on the interactions between Dvl1 and Wnt receptors, cells co-expressing EGFP-tagged D Fzd7 or E LRP6 and mCherry-tagged Dvl1 were used to perform FLIM-FRET. To examine the effect of oncogenic APC on plasma membrane Wnt receptor locali- zation, cells co-expressing EGFP-tagged F Fzd7 or G LRP6 and tH-RFP were used to perform FLIM-FRET analyses. For FLIM-FRET experiments, YAMC, IMCE, and IMCE

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R andDSystems,Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: In Vitro, Expressing, Clinical Proteomics, Membrane

Journal: World Journal of Gastrointestinal Surgery

Article Title: Effects of low-density lipoprotein cholesterol on lymph node metastasis after radical esophagectomy

doi: 10.4240/wjgs.v17.i8.106898

Figure Lengend Snippet: Gene expression profiling interactive analysis and tumor immune estimation resource version 2 analyses based on the cancer genome atlas database. A: Expression of low-density lipoprotein receptors in esophageal cancer (EC). The levels of LRP5 and LRP6 are higher in EC than those in normal esophageal tissues; B: LRP5 and LRP6 are correlated with a poor clinical prognosis. Overall survival curves of LRP5 and LRP6; C: Correlation between LRP6 and immune cell infiltration. LRP6 is positively correlated with B lymphocyte infiltration and negatively correlated with DC infiltration.

Article Snippet: After antigen retrieval, the tissue sections were incubated with the primary antibody against LRP6 [sc-25317 (dilution 1:50), Santa Cruz Biotech, Dallas, TX, United States] and CD20 [ab64088 (dilution 1:100), Abcam, Cambridge, MA, United States] at 4 °C overnight, followed by incubation with FITC-conjugated secondary antibody and counter-staining with DAPI.

Techniques: Gene Expression, Expressing

Journal: World Journal of Gastrointestinal Surgery

Article Title: Effects of low-density lipoprotein cholesterol on lymph node metastasis after radical esophagectomy

doi: 10.4240/wjgs.v17.i8.106898

Figure Lengend Snippet: Expression of LRP6 and infiltration of B lymphocytes in N+ and N0 group. A: Immunofluorescence of LRP6 and B lymphocytes in N+ and N0 group respectively; B: Quantitative analysis with Mann-Whitney U test of CD20. Infiltration of B lymphocytes are significantly higher in the N+ group; C: Quantitative analysis with Mann-Whitney U test of LRP6. Expression of LRP6 is significantly higher in the N+ group; D: Quantitative analysis with Mann-Whitney U test of Merg. Infiltration of LRP6+B lymphocytes are significantly higher in the N+ group.

Article Snippet: After antigen retrieval, the tissue sections were incubated with the primary antibody against LRP6 [sc-25317 (dilution 1:50), Santa Cruz Biotech, Dallas, TX, United States] and CD20 [ab64088 (dilution 1:100), Abcam, Cambridge, MA, United States] at 4 °C overnight, followed by incubation with FITC-conjugated secondary antibody and counter-staining with DAPI.

Techniques: Expressing, Immunofluorescence, MANN-WHITNEY

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 Knock Out screen identifies potential host factors that are essential for CDV-OP infectivity. (A) Schematic outline of the canine genome-wide CRISPR/Cas9 KO screen. OP neon , mNeonGreen expressing CDV-OP strain. (B) Infection of canine mammary carcinoma P114 cells with CDV-OP (left panel) or CDV wild-type (right panel) expressing mNeonGreen (OP neon and wt neon , respectively). (C) Assessment of cN4-expression in P114 cells by immunofluorescence (IF) analyses, using an anti-N4 antibody. (D) Investigation of cN4 (with GAPDH as a control) mRNA expression via reverse transcription-PCR. (E) Enrichment of cells acquiring CDV-OP-resistance through successive rounds of infection of P114 cells transduced with the canine genome-wide CRISPR library. (F) Cumulative distribution and area under the curve (AUC) analysis of the library representation. The representation was evaluated for the pDNA of the Beauty library (blue graph) as well as for the genomic DNA that was extracted from the D0 population (experiment 1 [E1], red/orange and experiment 2 [E2], green). An ideally distributed library is shown in black. The percentages indicate each library’s representation at 90% cumulative reads, and the AUC values are indicated in the legend. (G) Boxplot representing the log fold change (LFC) (D15, compared to the plasmid DNA of the library [pDNA], obtained for the control population) of genes known to be essential or nonessential. (H) Volcano plot representing the depleted (log fold change [LFC] < 0) and enriched (LFC > 0) genes after four rounds of infection with OP neon , compared to the control population. The LFC and P values were calculated from two independent replicates via a MAGeCK analysis. Each dot represents one gene for which at least two gRNAs (out of four) were used for the analysis. Selected hits are color-coded. (I) Schematic illustration of the LRP6 receptor, consisting of four β-propeller domains linked by epidermal growth factor (EGF)-like motives, low-density lipoprotein receptor (LDLR) class A repeats, a transmembrane domain, and a cytoplasmic tail.

Article Snippet: The plasmid (pCMV6) encoding a C-terminally Myc-DDK-tagged human LRP6 protein (hLRP6) was ordered from Origene ( NM_002336 ).

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Expressing, Immunofluorescence, Control, Reverse Transcription, Transduction, Plasmid Preparation

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: Ablation of LRP6 expression decreases CDV-OP infection in various canine cell lines. (A) Tracking of indels by decomposition (TIDE) analyses. Frequency of frameshift short insertions/deletions of LRP6 KO cells (green) using gRNA 1 or 2 (L1 and L2, respectively) or a nontargeting (NT) gRNA. In-frame mutations are color-coded in blue, and the wt genotype is color-coded in red. (B) Fluorescent syncytium formation of LRP6-expressing or ablated (LRP6 KO ) cells infected with OP neon . L1, LRP6 KO gRNA1; L2, LRP6 KO gRNA2; NT, nontargeting gRNA control. Pictures of stitched 96-wells were taken 3 days postinfection (dpi) (2 dpi for Vero cells). (C) Quantitative assessment of fluorescence emission from OP neon -infected cells, normalized to the NT. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the NT and those from the indicated KO cells (*, P < 0.05; ****, P < 0.0001). (D) The viral titers of OP neon in LRP6-expressing and LRP6 KO cells are displayed in Log 10 TCID 50 /mL. The means and standard deviations (SD) of data from six independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the NT and those from the indicated KO cells (**, P < 0.01; ****, P < 0.0001). (E) Assessment of LRP6 expression in the indicated cells via Western blotting analyses, using an anti-LRP6 antibody. As a loading control, actin was detected using a monoclonal anti-actin antibody. (F) Band intensities were recorded with the Bio-1D software. The means and standard deviations (SD) of data from three independent experiments, normalized to the NT, are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test using GraphPad Prism v.9 to analyze differences between the NT and the LRP6 KO values (***, P < 0.001; ****, P < 0.0001).

Article Snippet: The plasmid (pCMV6) encoding a C-terminally Myc-DDK-tagged human LRP6 protein (hLRP6) was ordered from Origene ( NM_002336 ).

Techniques: Expressing, Infection, Control, Fluorescence, Comparison, Western Blot, Software

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: A point mutation in the CDV-OP receptor-binding protein (A393T) prevents infectivity in Vero and P114 cells. (A) Assessment of LRP6-HA protein expression via Western blotting analyses, using an anti-HA antibody in P114-LRP6 KO cells (P114-L2) reconstituted with cLRP6-HA (P114-L2/L-HA) or not (P114-L2/empty). (B) IF staining of indicated cells, using a monoclonal anti-HA MAb. (C) Quantitative assessment of fluorescence emission from OP neon -infected cells, using an MOI of 1. Images were taken at 3 dpi. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using an unpaired t test, using GraphPad Prism v.9 to analyze the differences between the P114-L2/L-HA and P114-L2/empty values (****, P < 0.0001). (D) The viral titers of OP neon in P114-L2/L-HA and P114-L2/empty cells are displayed in Log 10 TCID 50 /mL. The means and standard deviations (SD) of data from six independent experiments are shown. Statistical significance was determined using an unpaired t test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the indicated cells (**, P < 0.01). (E) Cell-cell fusion induced by cells transiently expressing the indicated H-proteins together with FOP and GFP. Schematic representation of the various H-proteins with the amino acid numberings of the exchanged H-fragments are shown. OP-derived fragments are colored-coded in white, and wt-derived fragments are highlighted in gray. Images were taken 2 days posttransfection (dpt). (F) Cell-cell fusion induced by cells transiently expressing the indicated H-protein variants harboring single (or double) point mutation(s) together with FOP and GFP. Images were taken at 2 dpt. (G) Infection of various cell lines with OP neon carrying a single point mutation (A393T) in the H-protein (OP neon HOP A393T ). Images were taken at 2 or 3 dpi for the Vero or other cell lines, respectively. (H) Quantitative assessment of the fluorescence emission from the OP neon HOP A393T -infected cells. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the Vero-cSLAM cells and those from other cell types (****, P < 0.0001).

Article Snippet: The plasmid (pCMV6) encoding a C-terminally Myc-DDK-tagged human LRP6 protein (hLRP6) was ordered from Origene ( NM_002336 ).

Techniques: Mutagenesis, Binding Assay, Infection, Expressing, Western Blot, Staining, Fluorescence, Derivative Assay, Comparison

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: LRP6 acts as a functional receptor for CDV-OP. (A) Assessment of the binding activities of various solH-proteins at the cell surfaces of the indicated cells via immunofluorescence (IF) analyses, using an anti-TST monoclonal antibody. (B) Qualitative assessment of cell-cell fusion upon mixing two cell populations expressing the indicated H/F combinations, the human or canine LRP6 proteins (or empty vector control), the split nanoluciferase (nLuc) reporter proteins, and either GFP or RFP (see Materials and Methods for more details). Images of fluorescence emission were taken 24 h after cell mixing. (C) Quantitative assessment of the cell-to-cell fusion of mixed cell populations. Luminescence triggered (upon the addition of the substrate) by the reconstituted nLuc reporter protein, which was observed using a multiplate reader (Cytation 5 device, BioTek). The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the hLRP6- or cLRP6-transfected cells and those from the empty vector-transfected control cells. (***, P < 0.001; ns, nonsignificant). (D) Assessment of the binding activity of various TST-tagged, soluble H-protein constructs by mixing them with either an Fc-carrying soluble cLRP6 variant (solLRP6-Fc), an Fc-carrying soluble cSLAM protein (solSLAM.V-Fc), or without any proteins (for control experiments) via coimmunoprecipitation assays and Western blot analyses. Whereas protein G beads were used for the immunoprecipitation step, immunoprecipitated (IP) and coimmunoprecipitated (coIP) proteins were investigated via Western blotting analyses, using an anti-human-Fc or a mouse anti-TST antibody. (E) An ELISA of soluble proteins was used for the coIP experiments. (F) Infection assays in the indicated J3T cells were performed, using VSVΔG pseudotyped with standard CDV-OP glycoproteins (left) or a receptor-binding protein that was defective in LRP6-binding (HOP A393T ; right). The assay was performed in the presence of an F-protein inhibitor (3G), an anti-VSV-G antibody, or DMSO. Since the VSVΔG genome also encodes the firefly luciferase reporter protein, the viral infectivity was assessed indirectly via the recording of the firefly luciferase activity in infected cells at 24 h postinfection. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from DMSO-treated J3T-NT cells and those from the indicated J3T cells that were treated with inhibitors or DMSO (****, P < 0.0001; ns, nonsignificant).

Article Snippet: The plasmid (pCMV6) encoding a C-terminally Myc-DDK-tagged human LRP6 protein (hLRP6) was ordered from Origene ( NM_002336 ).

Techniques: Functional Assay, Binding Assay, Immunofluorescence, Expressing, Plasmid Preparation, Control, Fluorescence, Comparison, Transfection, Activity Assay, Construct, Variant Assay, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Infection, Luciferase

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: Oligonucleotides used for genome editing

Article Snippet: The plasmid (pCMV6) encoding a C-terminally Myc-DDK-tagged human LRP6 protein (hLRP6) was ordered from Origene ( NM_002336 ).

Techniques: Sequencing

Journal: mBio

Article Title: LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus

doi: 10.1128/mbio.03114-22

Figure Lengend Snippet: PCR primers used for TIDE analysis

Article Snippet: The plasmid (pCMV6) encoding a C-terminally Myc-DDK-tagged human LRP6 protein (hLRP6) was ordered from Origene ( NM_002336 ).

Techniques: Sequencing