Journal: Molecular Medicine Reports

Article Title: Dickkopf-3 upregulation mediates the cardioprotective effects of curcumin on chronic heart failure

doi: 10.3892/mmr.2018.8783

Figure Lengend Snippet: Effects of curcumin on DKK-3, p38, JNK and ASK1 protein expression levels. (A) Representative images of DKK-3 and (B) quantification of its expression levels in LVAW, as evaluated by immunohistochemical staining. (C) Representative images of DKK-3 and (D) quantification of its expression, as evaluated by western blot analysis. (E) Representative images of p-p38, p-JNK and p-ASK1 and quantification of (F) p-p38; (G) p-JNK and (H) p-ASK1 expression levels, as measured by western blot analysis. Magnification, ×200. Scale bar=50 µm. All data are expressed as the mean ± stadard deviation, n=10. *P<0.05. ASK1, apoptosis signal-regulating kinase 1; CHF, chronic heart failure; Con, control; Cur, curcumin; DKK-3, Dickkopf-related protein 3; LVAW, left ventricular posterior wall thickness; p, phosphorylated; T, total; p38, p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: Primary rabbit polyclonal anti-DKK-3 antibody (1:200; cat. on. bs-2686R; BIOSS) and secondary antibody mouse anti-rabbit IgM/HRP (1:200, bs-0369M-HRP, BIOSS) were used in the staining.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: p53-dependent upregulation of Wnt/β-catenin signalling antagonist Dkk3 is required for Cdc7-depletion-induced cell cycle arrest. (A) Cytoplasmatic protein fractions (CF) and crude nuclear extracts (NE) from untreated (UT), control-siRNA (CO) and CDC7-siRNA (Cdc7KD)-transfected IMR90 cells (72 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). (B) At the same time point, CO and Cdc7KD cells were fixed and β-catenin, Myc and cyclin D1 detected by indirect immunofluorescence using a fluorescein-labelled secondary antibody. (C, D) CF and NE samples prepared from CO, Cdc7KD and doubly depleted Cdc7/Dkk3 (Cdc7KD/Dkk3KD) cells 48 and 72 h post-transfection were analysed by immunoblotting with the indicated antibodies. (E) 72 h post-transfection CO, Cdc7KD and Cdc7KD/Dkk3KD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. Chromatin-bound PCNA and γH2A.X (inset: higher magnification) were detected by indirect immunofluorescence with anti-PCNA and anti-γH2A.X antibodies and a fluorescein-labelled secondary antibody. DNA was stained with propidium iodide (BrdU) or DAPI (PCNA and γH2A.X). Apoptotic cell death was detected in doubly depleted Cdc7KD/Dkk3KD cells by (F) phase contrast microscopy and by (G) immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: Immunodetection of Dkk3 was blocked by preincubation of Dkk3 antibody with recombinant human Dkk3 (1118-DK-050; R&D Systems Europe, Abingdon, UK) (1:1 w/w).

Techniques: Control, Transfection, Western Blot, Immunofluorescence, Staining, Microscopy

Journal: bioRxiv

Article Title: Wnt signaling drives stromal inflammation in inflammatory arthritis

doi: 10.1101/2025.01.06.631510

Figure Lengend Snippet: a , Simplified schematic of Wnt signaling illustrating the role of beta-catenin in the canonical pathway as well as calcium signaling and Rho/Rac signaling in the non-canonical pathway. b , tSNE plot of scRNA-seq data from disaggregated and flow-sorted RA and OA synovial tissue. c , Dot plot of data from b showing expression of Wnt pathway members among synovial populations. Individual clusters are grouped according to the labeled cell type. “Fibro” indicates fibroblasts and “Macro” indicates macrophages. For b and c , 5,265 sorted cells from n=14 RA and n=3 OA synovial tissue samples are shown. d , UMAP depicting fibroblast subpopulations in stromal-enriched scRNA-seq dataset sorted from RA and OA synovial biopsy samples. e , Expression of RSPO3, DKK3, THY1, and PRG4 in UMAP from d . Red arrows highlight areas of RSPO3 or DKK3 expression as indicated. f , Depiction of trajectory analysis to evaluate RSPO3-DKK3 gradient using a linear trajectory created connecting the centroids of expression of DKK3 and RSPO3 in Harmony-corrected PCA space from dataset in d and a pseudotime assigned to the trajectory. The expression of RSPO3 and DKK3 along this trajectory is depicted along with two genes, G0S2 and FMOD, which were identified by hierarchical clustering analysis to show a similar pattern of expression. g , Depiction of gene and pathway terms identified to be associated with RSPO3 or DKK3 in the trajectory analysis. For d-g , 47,579 cells are projected to UMAP or Harmony-corrected PCA space from OA (n=6) and RA (n=15) samples.

Article Snippet: Antibodies used for multiplex tissue immunofluorescence (IF) staining for RSPO3 (Clone 1E4F1, Proteintech, 1:100), VE-cadherin (Clone E6N7A, CST, 1:100) CD3 (Clone F7.2.38, DAKO, 1:200), Podoplanin/PDPN (EPR7072, Abcam, 1:100), CD90 (clone D3V8A, CST, 1:100), DKK3 (10365-1-AP, Proteintech, 1:200) and CD45 (clone D9M8I, CST, 1:100) was performed.

Techniques: Expressing, Labeling

Journal: bioRxiv

Article Title: Wnt signaling drives stromal inflammation in inflammatory arthritis

doi: 10.1101/2025.01.06.631510

Figure Lengend Snippet: a , tSNE plot showing expression of the indicated Wnt ligands from of scRNA-seq data of RA and OA synovial tissue. b , Dot plot showing expression of Wnt ligands among synovial populations using the dataset from ( a ). B1-B4 are B cell subsets, F1-F4 are fibroblast subsets, M1-M4 are monocyte subsets, and T1-T6 are T cell subsets. c , tSNE plot showing scRNA0seq from dataset from ( a ) showing expression of FZD7, FZD8, RSPO3, and DKK3. d , Bar graph of qPCR showing expression of AXIN2 in response to stimulation with the indicated Wnt ligands. STB-020 and RA190321 are individual RA cell lines (n=3 well replicates per condition). Mean ± SD is depicted. Significance was determined using a one-way ANOVA with Dunnett’s multiple comparisons test ****p<0.0001. In A-C, 5,265 sorted cells are shown with n=3 OA samples and n=14 RA samples.

Article Snippet: Antibodies used for multiplex tissue immunofluorescence (IF) staining for RSPO3 (Clone 1E4F1, Proteintech, 1:100), VE-cadherin (Clone E6N7A, CST, 1:100) CD3 (Clone F7.2.38, DAKO, 1:200), Podoplanin/PDPN (EPR7072, Abcam, 1:100), CD90 (clone D3V8A, CST, 1:100), DKK3 (10365-1-AP, Proteintech, 1:200) and CD45 (clone D9M8I, CST, 1:100) was performed.

Techniques: Expressing

Journal: bioRxiv

Article Title: Wnt signaling drives stromal inflammation in inflammatory arthritis

doi: 10.1101/2025.01.06.631510

Figure Lengend Snippet: a , Multispectral immunofluorescence staining of RA synovial tissue highlighting expression of RSPO3 (green), DKK3 (magenta), and DAPI (blue). Carets indicate fibroblasts with high expression of RSPO3 but low expression of DKK3 and arrows indicate fibroblasts with high expression of DKK3 and low expression of RSPO3. Scale bar, 20 μM. b , Extended view of multispectral immunofluorescence staining of RA synovial tissue shown in a . Box indicates area depicted in a . Scale bar, 100 μM.

Article Snippet: Antibodies used for multiplex tissue immunofluorescence (IF) staining for RSPO3 (Clone 1E4F1, Proteintech, 1:100), VE-cadherin (Clone E6N7A, CST, 1:100) CD3 (Clone F7.2.38, DAKO, 1:200), Podoplanin/PDPN (EPR7072, Abcam, 1:100), CD90 (clone D3V8A, CST, 1:100), DKK3 (10365-1-AP, Proteintech, 1:200) and CD45 (clone D9M8I, CST, 1:100) was performed.

Techniques: Immunofluorescence, Staining, Expressing

Journal: bioRxiv

Article Title: Wnt signaling drives stromal inflammation in inflammatory arthritis

doi: 10.1101/2025.01.06.631510

Figure Lengend Snippet: a , Harmony-corrected PCA plot of scRNA-seq data from 47,812 OA and RA synovial fibroblasts are shown with Wnt-5a activation scores, RSPO3 expression, and DKK3 expression depicted. Red arrows indicate areas of increased expression. b , Wnt-5a activation scores are shown for OA (n=11) and leukocyte-rich RA (n=16) synovial samples from bulk RNA sequencing data. c , RSPO3 and DKK3 enrichment scores are shown for OA (n=11) and leukocyte-rich RA (n=16) synovial samples from bulk RNA sequencing data collected by the Accelerated Medicines Partnership. d , Graph depicting correlation of Wnt-5a activation scores with RSPO3 (left) and DKK3 (right) enrichment scores for bulk RNA sequencing data of OA (n=11) and leukocyte-rich RA (n=16) synovial fibroblasts. e , Harmony-corrected PCA plot depicting RSPO3 and DKK3 expression in fibroblasts of the salivary gland from patients with Sjogren’s disease (n=7) or sicca symptoms (n=6), lung tissue from patients with ILD (n=19) or control (n=4), and colon from patients with inflammatory bowel disease (IBD) (n=8) or control (n=5) from samples collected by the Roche Fibroblast Network Consortium. f , On the left, a graph of Wnt-5a activation scores and RSPO3 enrichment scores are shown for gut fibroblast samples from healthy control patients and patients with IBD. On the right, Wnt-5a enrichment scores are shown for synovial fibroblast samples obtained from healthy control patients (n=5), non-inflamed IBD (n=4) and inflamed IBD (n=8).

Article Snippet: Antibodies used for multiplex tissue immunofluorescence (IF) staining for RSPO3 (Clone 1E4F1, Proteintech, 1:100), VE-cadherin (Clone E6N7A, CST, 1:100) CD3 (Clone F7.2.38, DAKO, 1:200), Podoplanin/PDPN (EPR7072, Abcam, 1:100), CD90 (clone D3V8A, CST, 1:100), DKK3 (10365-1-AP, Proteintech, 1:200) and CD45 (clone D9M8I, CST, 1:100) was performed.

Techniques: Activation Assay, Expressing, RNA Sequencing Assay, Control

Journal: Carcinogenesis

Article Title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

doi: 10.1093/carcin/bgr073

Figure Lengend Snippet: Fig. 1 MYCN knockdown increase DKK3 mRNA and secreted protein in neuroblastoma cells—(A) Real-time qRT–PCR measurements of DKK3 mRNA levels in SK-N-BE(2) and Kelly cells induced to knock down MYCN expression. Secreted endogenous DKK3 proteins from (B) SK-N-BE(2) and (C) Kelly cells induced to knock down MYCN expression and (D) SH-EP Tet21N cells induced to repress exogenous MYCN overexpression were measured with ELISA. Data presented are mean values ± SD shown as fold change compared with control that is normalized to 1. P , 0.05 versus respective control.

Article Snippet: Sections were incubated overnight at 4 C with a biotinylated goat anti-human DKK3 antibody (R&D Systems).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Control

Journal: Carcinogenesis

Article Title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

doi: 10.1093/carcin/bgr073

Figure Lengend Snippet: Fig. 2 DKK3 promoter methylation status in neuroblastoma tumors and cell lines—methylation-specific PCR was performed on bisulfite-treated DNA from neuroblastoma tumors and cell lines. Lanes labeled U and M contain PCR products amplified from primers recognizing unmethylated and methylated DKK3 promoters, respectively. All neuroblastoma samples investigated were unmethylated. DNA from the breast cancer cell lines MDA-MB231 and HS578t were used as methylated and unmethylated controls, respectively (25). NTC represents the no template control. MYCN- amplified samples are marked with filled squares.

Article Snippet: Sections were incubated overnight at 4 C with a biotinylated goat anti-human DKK3 antibody (R&D Systems).

Techniques: Methylation, Labeling, Control

Journal: Carcinogenesis

Article Title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

doi: 10.1093/carcin/bgr073

Figure Lengend Snippet: Fig. 3 Luciferase assays for mir-92a, mir-92b and let-7e—luciferase activity of HEK293 cells cotransfected with the wild-type (pMIR-DKK3 wt) or mutated DKK3-3#UTR luciferase vector and miRNA mimics of mir-92a (A), mir-92b (B) or let-7e (C). Mut mir-92 seed is mutated in the predicted mir-92 seed sequence at positions 28–29 in DKK3-3#UTR. Similarly, mut let-7 seed1, mut let-7 seed2 and mut let-7 seed1 þ 2 were mutated in the predicted let-7 seed sequences at positions 550–556, 180–185 and 550–556 þ 180–185 in DKK3-3#UTR, respectively. A plasmid constitutively expressing Renilla luciferase was used for normalization of the data. Data shown are mean values ± SD of the ratio of normalized luciferase activity in miRNA mimic and control transfections. Negative controls are normalized to 1. P , 0.05 versus respective control. NS—no significance.

Article Snippet: Sections were incubated overnight at 4 C with a biotinylated goat anti-human DKK3 antibody (R&D Systems).

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Sequencing, Expressing, Control, Transfection

Journal: Carcinogenesis

Article Title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

doi: 10.1093/carcin/bgr073

Figure Lengend Snippet: Fig. 4 DKK3 ELISA analyses of culture media from neuroblastoma cell lines treated with miRNA mimics and antagomirs—(A) Non-MNA neuroblastoma cell lines SH-SY-5Y and SK-N-AS were transfected with mir-92a, mir-92b, let-7e or negative control miRNA (mir-NC) mimics. (B) MNA neuroblastoma cell lines SK-N-BE(2) and Kelly were transfected with antagomir-92a (antimir-92) or a negative control antagomir (antimir-NC). Secretion of endogenous DKK3 proteins to the culture media was measured using an ELISA kit. Data shown are mean values ± SD of the ratio of DKK3 proteins secreted to the culture media normalized to total protein in cell extracts of miRNA mimic/antagomir and control transfections. Negative controls are normalized to 1. P , 0.05 compared with antimir-NC or mir-NC.

Article Snippet: Sections were incubated overnight at 4 C with a biotinylated goat anti-human DKK3 antibody (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Negative Control, Control

Journal: Carcinogenesis

Article Title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

doi: 10.1093/carcin/bgr073

Figure Lengend Snippet: Fig. 5 DKK3 and mir-92a expression correlation in neuroblastic tumors: visual representation of DKK3 mRNA and mir-92a miRNA expression in 95 neuroblastic tumors, ranked horizontally from left to right according to their DKK3 expression. DKK3 and mir-92a expression values for each tumor are visualized with red circles and blue rectangles, respectively: DKK3 and mir-92a show a highly significant negative correlation. Below the graph is the clinical annotation of the tumor samples. For expression value calculations and statistics, see Materials and Methods.

Article Snippet: Sections were incubated overnight at 4 C with a biotinylated goat anti-human DKK3 antibody (R&D Systems).

Techniques: Expressing

Journal: Carcinogenesis

Article Title: MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

doi: 10.1093/carcin/bgr073

Figure Lengend Snippet: Fig. 6 Immunohistochemical staining of DKK3 in neuroblastoma tumors—(A) A human neuroblastoma tissue section stained with a red fluorescently labeled (Alexa 594) anti-DKK3 monoclonal antibody, together with a green fluorescently labeled (Alexa 488) anti-CD31 monoclonal antibody. The nuclei were stained with 4#,6-diamidino-2-phenylindole, which are represented in blue. The merge represents an overlay view of the DKK3, CD31 and 4#,6-diamidino-2-phenylindole image; 600 magnification. (B) Immunohistochemical staining of DKK3 in neuroblastoma primary tumors and ganglioneuromas showing specific staining of DKK3 in tumor vasculature (left image, sample no. 8 in Supplementary Table 2, available at Carcinogenesis Online, 600 magnification) and in differentiated ganglion cells of a benign ganglioneuroma (right image, sample no. 28 in Supplementary Table 2, available at Carcinogenesis Online, 400 magnification).

Article Snippet: Sections were incubated overnight at 4 C with a biotinylated goat anti-human DKK3 antibody (R&D Systems).

Techniques: Immunohistochemical staining, Staining, Labeling