Journal: PLoS ONE

Article Title: Olive Oil and Vitamin D Synergistically Prevent Bone Loss in Mice

doi: 10.1371/journal.pone.0115817

Figure Lengend Snippet: Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ‡ p<0.05 vs VO-SH, # p<0.05 vs RO-OVX, $ p<0.05 vs VO-OVX, £ p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; ALP, alkaline phosphatase; OCN, osteocalcin; OPN, osteopontin; Col1a1, type I collagen; Lrp5, low density lipoprotein receptor-related protein 5; Sfrp1, secreted frizzled related sequence protein 1; Sost1, sclerostin; Esr1, estrogen receptor 1.

Article Snippet: Sequence references are: Gapdh;Gm12070;Gm10481-Mm03302249_g1, Trap (Acp5)-Mm00475698_m1, Alpl-Mm01187117_m1, Ocn (Bglap;Bglap-rs1;Bglap2)-Mm03413826_mH, Ccl2-Mm00441243_g1, Col1a1-Mm00801666_g1, Comp-Mm00489490_m1, Ctsk-Mm00484036_m1, Esr1-Mm00433147_m1, Il1b-Mm99999061_mH, Il6-Mm99999064_m1, Itgb3-Mm00443980_m1, Lrp5-Mm00493179_m1, Mmp2-Mm01253621_m1, Nos2-Mm00440488_m1, Pparg-Mm01184322_m1, Sfrp1-Mm03053883_s1, Sost-Mm03024247_g1, osterix (Sp7)-Mm00504574_m1, osteopontin (Spp1)-Mm00436767_m1, Tlr2-Mm00442346_m1, Tlr4-Mm00445273_m1, Rank (Tnfrsf11a)-Mm00437135_m1, Opg (Tnfrsf11b)-Mm01205928_m1, Rankl (Tnfsf11)-Mm00441908_m1.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Olive Oil and Vitamin D Synergistically Prevent Bone Loss in Mice

doi: 10.1371/journal.pone.0115817

Figure Lengend Snippet: The effect of ovariectomy is represented by dotted boxes, the arrow (before the gene name), showing the modification of gene expression. The effect of virgin olive oil and vitamin D3 is represented by the grey boxes, the arrow (after the gene name) showing the impact on gene expression. The arrows between the genes outline regulation pathways. ALP, alkaline phosphatase; CCl2, chemokine (C-C motif) ligand 2; Col1a1, type I collagen; Ctsk, catepsin K; Esr1, estrogen receptor 1; IL-1β, interleukin-1β; IL-6, interleukin-6; Itg-β3, β3-integrin; Lrp5, low density lipoprotein receptor-related protein 5; MMP-2, matrix metalloproteinase 2; Nos2, nitric oxide synthase; OCN, osteocalcin; OPN, osteopontin; Sost1, sclerostin; Tlr2, toll like receptor 2; Tlr4, toll like receptor 4; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Sequence references are: Gapdh;Gm12070;Gm10481-Mm03302249_g1, Trap (Acp5)-Mm00475698_m1, Alpl-Mm01187117_m1, Ocn (Bglap;Bglap-rs1;Bglap2)-Mm03413826_mH, Ccl2-Mm00441243_g1, Col1a1-Mm00801666_g1, Comp-Mm00489490_m1, Ctsk-Mm00484036_m1, Esr1-Mm00433147_m1, Il1b-Mm99999061_mH, Il6-Mm99999064_m1, Itgb3-Mm00443980_m1, Lrp5-Mm00493179_m1, Mmp2-Mm01253621_m1, Nos2-Mm00440488_m1, Pparg-Mm01184322_m1, Sfrp1-Mm03053883_s1, Sost-Mm03024247_g1, osterix (Sp7)-Mm00504574_m1, osteopontin (Spp1)-Mm00436767_m1, Tlr2-Mm00442346_m1, Tlr4-Mm00445273_m1, Rank (Tnfrsf11a)-Mm00437135_m1, Opg (Tnfrsf11b)-Mm01205928_m1, Rankl (Tnfsf11)-Mm00441908_m1.

Techniques: Modification, Gene Expression

Journal: Frontiers in pharmacology

Article Title: Sini San Inhibits Chronic Psychological Stress-Induced Breast Cancer Stemness by Suppressing Cortisol-Mediated GRP78 Activation.

doi: 10.3389/fphar.2021.714163

Figure Lengend Snippet: FIGURE 6 | SNS interrupts the interaction between GRP78 and LRP5 to suppress Wnt/β-catenin signaling. (A) After 4T1 cells were treated with cortisol for 24 h, the translocation of GRP78 to the cell membrane was monitored by immunofluorescence. Red: DiI-cell membrane tracker; green: GRP78. Co-localization of GRP78 and the cell membrane is shown as yellow fluorescence. (B) The 4T1 cells were treated with cortisol for 24 h, and the co-localization of GRP78 and LRP5 was detected by immunofluorescence. Co-localization is shown as yellow fluorescence. (C) The effect of SNS (200 μg/ml) on cortisol-induced cell membrane translocation of GRP78 was detected by immunofluorescence following SNS treatment for 24 h. (D) The effect of SNS (200 μg/ml) on the co-localization of GRP78 and LRP5 induced by cortisol was detected by immunofluorescence following SNS treatment for 24 h. (E) The expression of LRP5, p-LRP5, and β-catenin in the GRP78-overexpressing 4T1 cells was measured by western blots. (F) The 4T1 cells or GRP78-overexpressing 4T1 cells were treated with SNS (200 μg/ml) for 24 h, and the changes in cortisol- induced LRP5, p-LRP5, and β-catenin expression were detected by western blots. (G) The 4T1 cells were treated with SNS (200 μg/ml) for 24 h, and changes in the interaction of GRP78 with LRP5 were analyzed by Co-IP assays. Input represents the total protein extracts prepared without the antibody coupling resin. NC indicates the negative control prepared by adding quenching buffer to the antibody coupling resin. NS, not significant. The scale bars indicate 10 μm. One representative experiment of three independent experiments is displayed.

Article Snippet: The primary antibody included GRP78 antibody (11587-1-AP, Proteintech, Rosemont, IL, United States), LRP5 (24899-1- AP, Proteintech, Rosemont, IL, United States); Fluorescently labeled anti-rabbit Alexa Fluor 488 and Alexa Fluor 555 conjugated antibodies were used as secondary antibodies.

Techniques: Translocation Assay, Membrane, Expressing, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: Frontiers in pharmacology

Article Title: Sini San Inhibits Chronic Psychological Stress-Induced Breast Cancer Stemness by Suppressing Cortisol-Mediated GRP78 Activation.

doi: 10.3389/fphar.2021.714163

Figure Lengend Snippet: FIGURE 7 | SNS inhibits CUMS-activated Wnt/β-catenin signaling in primary and metastatic lesions of breast cancer in mice. (A) The expression of Ki-67 in primary and metastatic lesions of the 4T1 tumor-bearing mice was detected by immunohistochemistry, reflecting the proliferation of breast cancer cells in vivo (the scale bars indicate 50 μm). (B) The TUNEL assays were used to detect in situ apoptosis in primary and metastatic lesions of the 4T1 tumor-bearing mice (the scale bars indicate 10 μm). (C) Membrane expression of GRP78 in the primary and metastatic lesions of the 4T1 tumor-bearing mice was detected by immunofluorescence (the scale bars indicate 10 μm). Red: DiI-cell membrane tracker; green: GRP78. Co-localization of GRP78 and the cell membrane is shown as yellow fluorescence. (D) The expression levels of LRP5, p-LRP5, and β-catenin in the primary tumors were measured by immunohistochemistry (the scale bars indicate 50 μm). Data are represented as the mean value ±SD. One representative experiment of three independent experiments is displayed. One-way ANOVA and Bonferroni’s post hoc test were applied.

Article Snippet: The primary antibody included GRP78 antibody (11587-1-AP, Proteintech, Rosemont, IL, United States), LRP5 (24899-1- AP, Proteintech, Rosemont, IL, United States); Fluorescently labeled anti-rabbit Alexa Fluor 488 and Alexa Fluor 555 conjugated antibodies were used as secondary antibodies.

Techniques: Expressing, Immunohistochemistry, In Vivo, TUNEL Assay, In Situ, Membrane

Journal: Frontiers in pharmacology

Article Title: Sini San Inhibits Chronic Psychological Stress-Induced Breast Cancer Stemness by Suppressing Cortisol-Mediated GRP78 Activation.

doi: 10.3389/fphar.2021.714163

Figure Lengend Snippet: FIGURE 8 | SNS inhibits CUMS-induced lung metastasis and stemness of breast cancer. SNS interrupts the interaction between GRP78 and LRP5 on the cell surface, thus inhibiting the Wnt/β-catenin signaling of breast CSCs.

Article Snippet: The primary antibody included GRP78 antibody (11587-1-AP, Proteintech, Rosemont, IL, United States), LRP5 (24899-1- AP, Proteintech, Rosemont, IL, United States); Fluorescently labeled anti-rabbit Alexa Fluor 488 and Alexa Fluor 555 conjugated antibodies were used as secondary antibodies.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: MicroRNA-210 Regulates Cancer Cell Proliferation through Targeting Fibroblast Growth Factor Receptor-like 1 (FGFRL1)

doi: 10.1074/jbc.m110.170852

Figure Lengend Snippet: FIGURE 3. Identification of candidate target genes degraded by miR-210 in KYSE-170 cells. A, Venn diagram showing overlapping sets of genes whose expression was decreased 5-fold by transfection of miR-210 and computationally predicted target genes of miR-210. B, list of four potential miR-210 tar- get genes with a signal value of more than 50 in ncRNA-transfected cells upon microarray analysis. C–F, expression levels of the four potential miR-210 tar- get mRNAs assessed by qRT-PCR. The values are shown relative to the value obtained with ncRNA (n 3; *, p 0.01). G, Western blot analyses of FGFRL1, NDUFA4, GPR177, LRP5L, and -actin proteins.

Article Snippet: An anti-FGFRL1, NDUFA4, GPR177, or LRP5L antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (1:100) was used as the primary antibody, respectively, and a horseradish peroxidase-conjugated IgG antibody (1:5,000) was used as the secondary antibody.

Techniques: Expressing, Transfection, Microarray, Quantitative RT-PCR, Western Blot

Journal: Journal of Biological Chemistry

Article Title: MicroRNA-210 Regulates Cancer Cell Proliferation through Targeting Fibroblast Growth Factor Receptor-like 1 (FGFRL1)

doi: 10.1074/jbc.m110.170852

Figure Lengend Snippet: FIGURE 4. Identification of candidate target genes of miR-210 in ESCC. A–D, FGFRL1, NDUFA4, GPR177, and LRP5L expression levels in ESCCs divided into two groups on the basis of miR-210 expression levels were assessed by qRT-PCR. The values are shown relative to the value obtained for the miR-210-low group (n 41; **, p 0.05; *, p 0.01). E, clinicopathologic characteristics of 82 ESCCs divided into two groups (n 41) on the basis of FGFRL1 expression levels. F, plot of log10FGFRL1 relative expression intensity against log10miR-210 relative expression intensity. The line represents an approximated curve. The correlation coefficient (r) and the p value indicate the statistical significance of the negative correlation between the x and y variables. G, immunohisto- chemistry for FGFRL1 on ESCC and NEST. These sections were stained by anti-FGFRL1 antibody and by hematoxylin and eosin (H&E).

Article Snippet: An anti-FGFRL1, NDUFA4, GPR177, or LRP5L antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (1:100) was used as the primary antibody, respectively, and a horseradish peroxidase-conjugated IgG antibody (1:5,000) was used as the secondary antibody.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling

doi: 10.3389/fimmu.2024.1383113

Figure Lengend Snippet: The effect of PAM2 on WNT signaling components. Gene expression of Lrp5, Lrp6, Sost and Dkk1 analyzed in calvaria dissected one (A, B, E, F) and five days (C, D, G, H) after injection of PAM2 or vehicle (CTRL). In situ hybridization for expression of Sost five days after injection of vehicle (CTRL) (I, J) or PAM2 (K, L) . OB , old bone; NB , new bone. Scale bars; (I, K) = 200 μm, (J, L) = 100 μm. Data presented as individual values with mean ± SEM as vertical lines. Statistical analyses were performed using unpaired Student’s t test. * P <0.05 vs CTRL, ** P <0.01 vs CTRL, *** P <0.001 vs CTRL, ns, non-significant.

Article Snippet: The following predesigned real-time PCR assays from Applied Biosystems were used for gene expression assays: Alpl (Mm00475834_m1), Col1a1 (Mm00801666_g1), Bglap (Mm01741771_g1), Runx2 (Mm00501580_m1), Sp7 ( Osterix ; Mm04209856_m1), Lrp5 (Mm01227476_m1), Lrp6 (Mm00999795_m1), Dkk1 (Mm00438422_m1), Sost (Mm00470479_m1), Wnt1 (Mm01300555_g1), Wnt3a (Mm00437337_m1), Wnt4 (Mm01194003_m1), Wnt5a Mm00437347_m1), Wnt7b (Mm01301717_m1), Wnt10b (Mm00442104_m1), Wnt16 (Mm00446420_m1), Tnfs11 ( Rankl ; Mm00441908_m1), Tnfrsf11b ( Opg ; Mm00435452_m1), Ctsk (Mm00484036_m1), Acp5 ( Trap ; Mm00475698_m1).

Techniques: Gene Expression, Injection, In Situ Hybridization, Expressing

Journal: Carcinogenesis

Article Title: Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic β-catenin levels to modulate Wnt signaling and intestinal homeostasis

doi: 10.1093/carcin/bgz007

Figure Lengend Snippet: BVES interacts with LRP6 and LRP5. (A) Reciprocal co-immunoprecipitation of Flag-tagged BVES (Flag-BVES) and GFP-tagged LRP6 (GFP-LRP6). HEK293T cells were transiently transfected with 4 μg of each plasmid using polyethylenimine. Filler plasmid was used to maintain equal DNA quantities. GFP-LRP6 was immunoprecipitated with GFP-binding protein magnetic beads following by immunoblotting with anti-Flag antibodies. Flag agarose resin was used to immunoprecipitate BVES followed by immunoblotting with anti-GFP antibodies. (B) Immunoprecipitation of Flag-BVES followed by immunoblotting for endogenous LRP5 and LRP6. 4 μg of vector or Flag-BVES was transfected into HEK293T cells using polyethylenimine. The LRP5 and LRP6 blots were developed using enhanced chemiluminescence and film, whereas the rest of the blots were immunoblotted using Odyssey infrared reagents as discussed in Materials and methods. (C) Schematic of the N-terminal myc-tag LRP6 (Myc-LRP6ICD) construct that spans amino acids 1364–1539 and contains the LRP6 transmembrane domain (TM) and proximal intracellular domain (ICD). Myc-LRP6ICD was transfected into HEK293T cells along with Flag-BVES as in (A) and immunoprecipitation experiments performed 48 h later. (D) Cells were transfected with 4 μg of Flag-BVES and then 48 h later treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 2 h before flag immunoprecipitation.

Article Snippet: Lysates were transferred to a 0.2 μm Nitrocellulose membrane (PerkinElmer) and then blocked using Odyssey Tris-buffered saline blocking buffer (LI-COR) for 30 min. Membranes were probed overnight at 4°C with the following antibodies diluted in Odyssey blocking buffer with 0.1% Tween-20: β-catenin (1:2000, BD Biosciences #610153), LRP6 (1:1000, Cell Signaling Technologies #3395), phospho-LRP6 Ser1490 (1:750, Cell Signaling Technologies #2568), LRP5 (1:1000, Cell Signaling Technologies #5731), DVL-2 (1:1000, Cell Signaling Technologies #3224), DVL-3 (1:1000, Cell Signaling Technologies #5731), Flag (1:1000, Sigma–Aldrich #F1804), GFP (1:1000, Cell Signaling Technologies #2956), β-Actin (1:5000, Sigma A5441) and GAPDH (1:4000, Cell Signaling Technologies #5174).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Magnetic Beads, Western Blot, Construct

Journal: Nature Communications

Article Title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

doi: 10.1038/s41467-025-60096-7

Figure Lengend Snippet: A Architecture of LRP-Venus and FZD-Nluc constructs. SP, signal peptide; FLAG, FLAG-tag; E1-E4, extracellular YWTD β-propeller/EGF domain repeats 1-4; LDL-A, low-density lipoprotein receptor type A repeats; TM, transmembrane domain; ICD, intracellular domain; CRD, cysteine-rich domain; 7TM, seven-transmembrane domain; C-term., receptor C-terminal domain. B Schematic depiction of the assay principle. Parts of this figure were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDrivs 4.0 International license. Created in BioRender. Voss, J. https://BioRender.com/lfyvjb8 . C , D . WNT-3A stimulation (1000 ng/ml) kinetics of HEK293 ( C ) or HEK293T ΔLRP5/6 ( D ) cells transfected with FZD 5 -Nluc and LRP5/6-Venus (yellow and blue circles, respectively; note: y-axes are not scaling equal in all figure panels). E Stimulation of HEK293 cells transfected with FZD 5 -Nluc and LRP5- or LRP6-Venus with 1 nM of WNT surrogate. F WNT-3A stimulation (1000 ng/ml) of HEK293 cells transfected with FZD 5 -Nluc and chimeric LRP5/6-Venus variants. G Architecture of LRP6 and LRP6-5A-Venus constructs. PPP(S/T)P/PPPAP, phosphorylation motifs. H. WNT-3A stimulation kinetics of HEK293A cells transfected with FZD 5 -Nluc and LRP6-/LRP6-5A-Venus (blue and light blue circles, respectively). Data are presented as mea n ± standard error of the mean (SEM) of five ( C ), four ( D ), or three ( E , F , H ) individual experiments, each performed in triplicate.

Article Snippet: The template sequences for LRP5 and LRP6 were obtained from Addgene (#115907 and #27282, respectively) , .

Techniques: Construct, FLAG-tag, Transfection, Phospho-proteomics

Journal: Nature Communications

Article Title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

doi: 10.1038/s41467-025-60096-7

Figure Lengend Snippet: A Schematic depiction of mutational paradigms. The FZD 5 R 6.32 A mutants preferentially signals in DVL-independent fashion. The DVL2 M2/M4 mutant cannot polymerize by its DIX domains. The DVL2 L445E mutant cannot engage FZD via its DEP domain. Created in BioRender. Voss, J. (2025) https://BioRender.com/zfu7r84 . B ΔBRET traces of HEK293 cells transfected either with WT FZD 5 -Nluc or FZD 5 R 6.32 A-Nluc (dark and light blue circles, respectively) and LRP6-Venus, stimulated with 1000 ng/ml WNT-3A. C ΔBRET traces of HEK293 and HEK293T ΔDVL1-3 cells, transfected with FZD 5 -Nluc, LRP6-Venus and either pcDNA (green and black circles), DVL2 (cyan and magenta), or DVL2-M2/M4 (dark cyan and purple) as indicated, stimulated with 1000 ng/ml WNT-3A. D ΔBRET traces of ΔDVL1-3 cells, transfected with FZD 5 -Nluc, LRP6-Venus and either pcDNA (black), DVL1 (green), DVL2 (magenta), or DVL3 (purple) as indicated, stimulated with 1000 ng/ml WNT-3A. E ΔBRET traces of ΔDVL1-3 cells, transfected with FZD 5 -Nluc, LRP6-Venus and either pcDNA (black), DVL2 (magenta), DVL2-M2/M4 (purple), DVL2-L445E (orange), or DVL2-M2/M4-L445E (red) as indicated, stimulated with 1000 ng/ml WNT-3A. F Scheme illustrating a proposed mechanism of DVL modulation of WNT-induced FZD 5 -LRP6 interaction. Created in BioRender. Voss, J. (2025) https://BioRender.com/3gct1ez . Data are shown as mea n ± SEM of three (B – FZD 5 R 6.32 A, C – data with DVL2-M2/M4 co-expression D, E) or four (B – wt FZD 5 , C – all other data) individual experiments performed in triplicate. Parts of this figure were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDrivs 4.0 International license.

Article Snippet: The template sequences for LRP5 and LRP6 were obtained from Addgene (#115907 and #27282, respectively) , .

Techniques: Mutagenesis, Transfection, Expressing

Journal: Nature Communications

Article Title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

doi: 10.1038/s41467-025-60096-7

Figure Lengend Snippet: A–D Kinetic ΔBRET traces of 1000 ng/ml WNT-3A ( A ), -5A ( B ), -10B ( C ), and -16B ( D ) at FZD 4,5,7 -Nluc (purple, red, and green, respectively) and LRP6-Venus. E Immunoblotting of phospho-LRP6, β-catenin, DVL2 and GAPDH (loading control) from whole cell lysates of HEK cells, stimulated for 2 h with 300 ng/ml of WNT-3A, -5A, -10B, and -16B or 1 nM surrogate WNT. The vehicle control (VC) was treated with HBSS and a CHAPS/EDTA mixture corresponding to that present in WNT preparations. F , G Densitometric analysis ( F , ratio of phospho-LRP6 (P-LRP6)/GAPDH; G , ratio of phosphorylated and shifted (PS-DVL2/DVL2) of blots shown in E. VC – black circle outlines, WNT-3A – blue, WNT-5A – red, WNT-10B – dark teal, WNT-16B –purple, surrogate WNT – gray. H . TOPFlash reporter gene assays in HEK293 and HEK293T ΔFZD 1-10 cells overexpressing FZD 4/5/7 stimulated with diverse WNTs (300 ng/ml for 24 h) or WNT surrogate (1 nM for 24 h). The TOPFlash ratio is given as a fold-increase over vehicle control; statistical analysis was performed with one-way-ANOVA versus vehicle control for each transfection condition. WNT-3A – blue, WNT-5A – red, WNT-10B – dark teal, WNT-16B –purple, surrogate WNT – gray. I . TOPFlash reporter gene assays in HEK293 cells stimulated with R-spondin 1 (RSPO1; 100 ng/ml), WNT-3A (300 ng/ml), WNT-16B (300 ng/ml), and WNT-R-spondin 1 combinations for 24 h. Vehicle – black circle outlines, R-spondin 1 – blue-gray, WNT-3A – blue, WNT-16B – purple, WNT-3A + R-spondin 1 – light blue, WNT-16B + R-spondin 1 – pink. Statistical significance was assessed by a one-way ANOVA using Dunnett’s post hoc test for multiple comparisons against the vehicle control (reporter gene assay data was log 10 -transformed prior to statistical analysis). Data points are shown as mea n ± SEM of three individual experiments ( n = 4 in Fid. 2 C, D for FZD 5 data and in Fig. for FZD 7 TOPFlash data), performed in triplicate in case of BRET assays and reporter gene assays. Exact p-values are detailed in the source data file.

Article Snippet: The template sequences for LRP5 and LRP6 were obtained from Addgene (#115907 and #27282, respectively) , .

Techniques: Western Blot, Control, Transfection, Reporter Gene Assay, Transformation Assay

Journal: Nature Communications

Article Title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

doi: 10.1038/s41467-025-60096-7

Figure Lengend Snippet: A . Representative single-molecule images showing SNAP-FZD 5 molecules, labeled with SNAP SiR-647 (left) and Halo-LRP6 molecules, labeled with Halo R110 (right). B . Single-molecule trajectory traces of SNAP-FZD 5 (magenta) and Halo-LRP6 (green). C . Proportion of molecular confinement at basal and following 100 nM WNT-3A and 100 nM WNT-16B stimulations. Basal – black, WNT-3A early – light blue, WNT-3A late – blue, WNT-16B early – light purple, WNT-16B late – purple. D . Estimated k on (left) and k off (right) values of FZD 5 -LRP6 interactions at basal and following 100 nM WNT-3A and 100 nM WNT-16B stimulations. Basal – black, WNT-3A early – light blue, WNT-3A late – blue, WNT-16B early – light purple, WNT-16B late – purple. E . Distributions of co-diffusion (blue) and co-confinement (red) events at basal and 100 nM WNT-3A and 100 nM WNT-16B stimulated conditions. F . Representative, dual-color single-molecule images showing SNAP-FZD 5 molecules, labeled with SNAP SiR-647 (magenta) and Halo-LRP6 molecules, labeled with Halo R110 (green) following 100 nM WNT-3A and 100 nM WNT-16B stimulations at late time-point. G . Cluster analysis showing the distributions of photobleaching steps following 100 nM WNT-3A and 100 nM WNT-16B stimulations at late time-point. (C-E) Data points are shown as median ± 95% confidence interval. Early stimulation: 2-10 min, late stimulation: 11-25 min. Statistical comparisons were made by Kruskal-Wallis followed by Dunn’s multiple comparison test. n = 18, 25, 22, 17, 27, 25 cells for FZD 5 -LRP6 basal, WNT-3A early, WNT-3A late, WNT-16B early, WNT-16B late, and β 2 AR-LRP6, respectively, from six independent experiments. See also Supp. Movies - . Exact p-values are detailed in the source data file.

Article Snippet: The template sequences for LRP5 and LRP6 were obtained from Addgene (#115907 and #27282, respectively) , .

Techniques: Labeling, Diffusion-based Assay, Comparison

Journal: Nature Communications

Article Title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

doi: 10.1038/s41467-025-60096-7

Figure Lengend Snippet: A Proportion of SNAP-FZD 5 and Halo-LRP6-5A molecular confinement at basal (black) and following 100 nM WNT-3A (early – light blue, late – dark blue) stimulation. B Representative, dual-color single-molecule image showing SNAP-FZD 5 molecules, labeled with SNAP SiR-647 (magenta) and Halo-LRP6-5A molecules, labeled with Halo R110 (green) following 100 nM WNT-3A stimulation at late time-point. C . Estimated kon (left) and koff (right) values of FZD 5 -LRP6-5A interactions at basal and following 100 nM WNT-3A stimulation (early – light blue, late – dark blue). D Distributions of co-diffusion (blue) and co-confinement (red) events at basal and 100 nM WNT-3A stimulated conditions. E Cluster analysis showing the distributions of photobleaching steps following 100 nM WNT-3A stimulation at late time-point. A , C , D Data points are shown as median ± 95% confidence interval. Early stimulation: 2-10 min, late stimulation: 11-25 min. Statistical comparisons were made by Kruskal-Wallis followed by Dunn’s multiple comparison test ( A , D ) and Mann-Whitney test C . n = 21, 26, 32 cells for FZD 5 -LRP6-5A basal, WNT-3A early, WNT-3A late, respectively, from three independent experiments. See also Supp. Movies – . Exact p-values are detailed in the source data file.

Article Snippet: The template sequences for LRP5 and LRP6 were obtained from Addgene (#115907 and #27282, respectively) , .

Techniques: Labeling, Diffusion-based Assay, Comparison, MANN-WHITNEY

Journal: Nature Communications

Article Title: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

doi: 10.1038/s41467-025-60096-7

Figure Lengend Snippet: A Direct comparison of the effects of WNT-3A and WNT-16B as probed in this study. B Two-step model of signal initiation and specification in WNT/β-catenin signaling. In a first step, WNT binding leads to ligand-induced association of FZD 5 and LRP6 (signal initiation) for both WNT-3A (top, red) and WNT−16B (bottom, blue). Upon higher-order receptor clustering and LRP6 phosphorylation, the signal is specified into WNT/β-catenin signaling (top, red). If these hallmarks are not met, the FZD-WNT complex can signal via other signaling pathways (bottom, blue). It is unclear whether LRP6 is involved in signal specification in that case. Created in BioRender. Voss, J. (2025) https://BioRender.com/e80oa99 . Parts of this figure were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDrivs 4.0 International license.

Article Snippet: The template sequences for LRP5 and LRP6 were obtained from Addgene (#115907 and #27282, respectively) , .

Techniques: Comparison, Binding Assay, Phospho-proteomics, Protein-Protein interactions

Journal: Journal of medical primatology

Article Title: The nonhuman primate kidney transcriptome in fetal development

doi: 10.1111/jmp.12340

Figure Lengend Snippet: Gene expression profiles for MAPK1, TP53, CCNG1, SMAD4, EIF4E and LRP5 are shown. The left graph shows the array data, and the right graph shows the QRT-PCR data. For gene array graphs, relative expression of log2-transformed values are shown. For QRT-PCR graphs, RQ values are shown on the y-axis. The time points are shown on the x-axis. Females are shown with white bars and males with hashed bars. The * denotes p < 0.05 for comparison with 90d for females and § denotes p<0.05 and Ψ denotes p=0.06 for comparison with 90d for males. For 60d, n=3, 1 male, 2 undetermined; 90d, 140d, 160d, 175d, PN and Adult, n=3 females and n=3 males per group; and 125d n=2 females and n=3 males per group.

Article Snippet: Gene-specific primers for CCNB1 , MAPK1 , TP53 , SMAD4 , EIF4e , and LRP5 were provided by the manufacturer (CCNB1, Hs00259126_m1; MAPK1, Hs01052196_m1; TP53, Hs01034253_m1; SMAD4, Hs00232068_m1; EIF4e, Hs00913390_m1; and LRP5, Hs00182031_m1).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Transformation Assay, Comparison