Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Dickkopf-3 (Dkk3) is protective against ischemic neuronal death. (A) Nissl staining in 10 μm coronal mouse brain sections collected every 320 μm along the rostro-caudal extension of the ischemic lesion in wild-type and Dkk3 knockout mice 1 day after middle cerebral artery occlusion (MCAO). (B) Infarct size in wild-type and Dkk3 knockout mice 1 day, 3 days or 7 days following MCAO. Values are means + SEM. * p < 0.05 vs. wild-type mice (Student’s t -test; day 1: n = 7–9, p = 0.0073, t (14) = 3.135; day 3: n = 4–5, p = 0.0143, t (7) = 3.24; day 7: n = 4–7, p = 0.0387, t (9) = 2.42). (C) Immunohistochemical analysis of Dkk3 in the cerebral cortex of wild-type mice at 6 h and 12 h following MCAO. Low and high magnification images are shown. (D) Double fluorescence immunostaining of Dkk3 and GFAP in the ischemic cortex of wild-type mice 12 h following MCAO. Arrowheads show Dkk3 expression in astrocytes. Arrows show Dkk3 expression in GFAP-negative cells.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Staining, Knock-Out, Immunohistochemical staining, Fluorescence, Immunostaining, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Protective activity of Dkk3 in cultured astrocytes exposed to oxidative damage or glucose deprivation. (A) Flow cytometry analysis (FACS) of live cells in cultured cortical astrocytes from wild-type and Dkk3 knockout mice (C57BL/6J strain) following 1 h of incubation with H 2 O 2 (250 μM). Values are means + SEM ( n = 3–4 per group). p < 0.05 vs. the respective control cultures not exposed to H 2 O 2 (*), or vs. wild-type cultures exposed to H 2 O 2 ( # two-way ANOVA + Fisher’s least significant difference (LSD); genotype, p < 0.001, F (1,11) = 15.79; treatment, p < 0.001, F (1,11) = 28.24; genotype × treatment, F (1,11) = 23.43). (B) Same as in (A) but after exposure to 4 h of glucose deprivation. Values are means + SEM. ( n = 6–9 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,25) = 26.73; treatment, p < 0.001, F (1,25) = 19.33; genotype × treatment, F (1,25) = 18.26). Dkk3 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (C) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p = 0.003, F (1,12) = 8.18; treatment, p = 0.004, F (1,12) = 6.53; genotype × treatment, F (1,12) = 5.82). VEGF protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (D) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,12) = 38.54; treatment, p = 0.003, F (1,12) = 6.60; genotype × treatment, F (1,12) = 6.90). Phospho-AKT protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (E) . Values are means + SEM ( n = 6 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,20) = 19.40; treatment, p = 0.016, F (1,20) = 3.09; genotype × treatment, F (1,20) = 3.93). Bcl2 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in (F) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. the respective cultures not exposed to glucose deprivation (*) or vs. wild-type cultures exposed to glucose deprivation ( # two-way ANOVA + Fisher’s LSD; genotype, p < 0.001, F (1,12) = 25.46; treatment, p = 0.038, F (1,12) = 1.74; genotype × treatment, F (1,12) = 3.93). VEGF protein levels in the whole brain of wild-type and Dkk3 knockout mice are shown in (G) . Values are means + SEM ( n = 3–4 per group). * p < 0.05 vs. wild-type mice (Student’s t-test ; p = 0.0005, t 5 = 7.98). Dkk3 and VEGF protein levels in the whole brain of CD1 and C57BL/6J mice are shown in (H) . Values are means ± SEM ( n = 3 per group). * p < 0.05 vs. CD1 mice (Student’s t-test ; Dkk3, p = 0.0099, t (4) = 4.62; VEGF, p = 0.0384, t (4) = 3.04). (I) FACS analysis of live cells in cultures of cortical astrocytes from CD1 mice incubated for 12 h with hrDkk3 (10 ng/ml) prior to a 1 h-exposure to H 2 O 2 (100 μM). Values are means + SEM ( n = 3 per group). p < 0.05 vs. the respective control cultures not exposed to H 2 O 2 (* p = 0.0001), or vs. the respective cultures not treated with hrDkk3 ( # p = 0.0013; one-way ANOVA + Fisher’s LSD; F (3,8) = 26.2). VEGF protein levels in lysates of cultured astrocytes from CD1 mice treated with hrDkk3 (10 ng/ml) for 12 h are shown in (J) . Values are means ± SEM ( n = 3 per group). * p < 0.05 vs. controls (Student’s t-test ; p = 0.0119, t (4) = 4.38). Ctrl = untreated controls.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Knock-Out, Incubation, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Dkk3 is protective against excitotoxic neuronal death in mixed cortical cultures. Neuronal death in mixed cortical cultures prepared from different mouse genotypes (all C57BL/6J strain) and challenged with 100 μM N -methyl-D-aspartate (NMDA) is shown in (A) . Values are means + SEM ( n = 4–8 per group). p < 0.05 vs. the respective cultures not treated with NMDA (*), or vs. cultures treated with NMDA from wild-type mice in both neurons astrocytes and neurons ( # two-way ANOVA + Fisher’s LSD; genotype, p = 0.002 for astrocytes knockout/neurons knockout; p = 0.026 for astrocytes knockout/neurons wild-type, F (3,40) = 2.06; treatment, p < 0.001 for astrocytes wild-type/neurons wild-type, astrocytes knockout/neurons wild-type and astrocytes knockout/neurons knockout; p = 0.003 for astrocytes wild-type/neurons knockout, F (1,40) = 77.52; genotype × treatment, F (3,40) = 2.07). Neuronal death in cultures from CD1 mice challenged with 45 μM NMDA followed or not by 20-h exposure to hrDkk3 (10 ng/ml) is shown in (B) . Values are means + SEM ( n = 4 per group). p < 0.05 vs. cultures not treated with NMDA (* p < 0.0001) or vs. cultures challenged with NMDA not exposed to hrDkk3 ( p = 0.0009; one-way ANOVA + Fisher’s LSD; F (3,12) = 83.69). Data obtained in cultures from CD1 mice challenged with 80 μM NMDA followed or not by 20-h exposure to hrDkk3 (10 ng/ml) w/wo ZM3238881 (10 μM) is shown in (C) . Values are means + SEM ( n = 5–8 per group). p < 0.05 vs. cultures not treated with NMDA (* p = 0.0005 for NMDA, p < 0.0001 for NMDA+ hrDkk3 + ZM323881, p = 0.0012 for NMDA+ ZM323881) or vs. cultures challenged with NMDA and not exposed to hrDkk3 ( # p = 0.0045; one-way ANOVA + Fisher’s LSD; F (5,32) = 8.39). Neuroprotection by hrVEGF against NMDA toxicity (here, 150 μM) in mixed cortical cultures from CD1 mice is shown in (D) . Cells were exposed to hrVEGF for 20 h following the NMDA pulse. Values are means + SEM ( n = 4–11 per group). p < 0.05 vs. cultures not treated with NMDA (* p < 0.0001) or vs. cultures challenged with NMDA not exposed to hrVEGF ( # p < 0.0001; one-way ANOVA + Fisher’s LSD; F (5,31) = 22.26).

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Knock-Out

Journal: Frontiers in Cellular Neuroscience

Article Title: Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor

doi: 10.3389/fncel.2018.00292

Figure Lengend Snippet: Schematic representation of the protective feedback Dkk3-mediated in astrocytes and/or neurons exposed to cellular stress (glucose deprivation, oxidative stress or ischemia). Dkk3 leads to VEGF upregulation with ensuing VEGFR2 activation, AKT phosphorylation and upregulation of the antiapoptotic factor Bcl2.

Article Snippet: For Dkk3 expression analysis, slices were incubated overnight with a goat polyclonal anti-Dkk3 antibody (1:50, RD System) and then for 1 h at room temperature (RT) with a secondary biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Activation Assay, Phospho-proteomics

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

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used for immunofluorescence were: mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), Rabbit Anti-Aβ (6E10 clone, chimeric, 1:1000, Novus Biologicals, Cat# NBP2-62566, RRID: AB_2917960 ), chicken anti-Bassoon (1:1,000, Synaptic Systems, Cat# 141 016, RRID: AB_2661779 ), Goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), Rabbit anti-Gephyrin (1:500, Synaptic Systems, Cat# 147 002, RRID: AB_2619838 ), Chicken anti-Glial Fibrillary Acidic Protein (GFAP) (1:500, Millipore, Cat# AB5541, RRID: AB_177521 ), Rabbit anti-Homer1 (1:1000, Synaptic Systems, Cat# 160 003, RRID: AB_887730 ), chicken Anti-GFP (1:500, Millipore, Cat# 06–896, RRID: AB_310288 ), rabbit anti-Iba1 (1:1000, FUJIFILM Wako Shibayagi, Cat# 019–1974,1 RRID: AB_839504 ), rat anti-LAMP1 (1:250, DSHB, 1D4B, RRID: AB_528127 ), rabbit Anti-NeuN (D3S3I) (1:1000, Cell Signaling Technology, Cat# 12943, RRID: AB_2630395 ), rabbit Anti-Neurofilament Heavy (1:5000, Abcam, Cat# ab8135, RRID: AB_306298 ), mouse Anti-PSD-95 (7E3-1B8) (1:500, Thermo Fisher Scientific, Cat# MA1-046, RRID: AB_2092361 ), guinea pig anti-vesicular GABA Transporter (vGAT) (1:500, Synaptic Systems, Cat# 131 004, RRID: AB_887873 ), guinea pig Anti-Vesicular Glutamate Transporter 1 (vGLUT1) (1:2000, Millipore, Cat# AB5905, RRID: AB_2301751 ), goat anti-Wnt7a/b (1:1000, R and D Systems, Cat# AF3460, RRID: AB_2304437 ), mouse Anti- beta Catenin (1:1000, BD Biosciences, Cat# 610153, RRID: AB_397554 ).

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Dkk3 does not influence T-cell development and activation in naïve Dkk3-deficient mice . (A) Thymocytes from naïve Dkk3 −/− mice and littermate controls were stained for CD4 and CD8 and analyzed by flow cytometry. Distribution of thymocyte subsets (DN: CD4 − CD8 − double negative; DP: CD4 + CD8 + double positive) is shown in a representative dot plot (left) and cumulative data of percentages of whole thymocytes (right; n = 4–5). (B) Absolute numbers of respective thymocyte subsets from (A) , determined by flow cytometry ( n = 4–5). (C) Flow cytometric analysis of CD69 cell surface expression on CD4 + , CD8 + , and CD4 + CD8 + double positive (DP) subsets from (A) . One representative histogram is shown ( n = 4–5). (D) Representative dot plot of CD25 + Foxp3 + cells among CD4 single positive cells in the thymus of Dkk3 −/− and WT control mice. (E) Analysis of CD4 + and CD8 + T-cell subsets, among CD3 positive cells, in the spleen of Dkk3 −/− mice compared to littermate controls. One representative dot plot (left) as well as respective cumulative data is shown. (F) Absolute numbers of lymphocyte subsets from (D) , determined by flow cytometry ( n = 4–5). (G) Analysis of CD4 + and CD8 + T-cell subsets among CD3 positive cells in peripheral lymph nodes (LN) of Dkk3 −/− mice and WT controls. One representative dot plot (left) as well as respective cumulative data is shown. (H) Absolute numbers of lymphocyte subsets from (F) , determined by flow cytometry ( n = 4–5). (I+K) Flow cytometric analysis of surface expression of the activation markers CD25 and CD69 on CD4 + (upper row) and CD8 + (lower row) T cells in (I) spleen and (K) peripheral lymph nodes of naïve Dkk3 −/− (red) and WT control (black) mice. Shown is one representative histogram ( n = 10). (J+L) Flow cytometric analysis of the surface expression of CD44 and CD62L on CD4 + (upper row) and CD8 + (lower row) T cells in (J) spleen and (L) peripheral lymph nodes of naïve Dkk3 −/− and WT control mice. Shown is one representative dot plot ( n = 8).

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Activation Assay, Staining, Flow Cytometry, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Dkk3 protects MHC class-I mismatched transplanted embryoid bodies from rejection . (A) Male CBK mice-derived embryoid bodies were transplanted under the kidney capsule of male CBA mice. Twenty-eight days after transplantation, remaining embryoid body tissue was stained with anti-Dkk3 antibody. Shown is one representative staining of accepted and remaining elements of the rejected tissue ( n = 3). (B) Male CBK mice-derived embryoid bodies were transplanted under the kidney capsule of male CBA mice. Seven days later, 1 mg anti-Dkk3 or isotype control antibody was administered, followed by 0.5 mg every fourth day. Twenty-eight days after transplantation, the acceptance rate of the transplants was determined (Log-rank test, n = 24, * p < 0.05).

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Derivative Assay, Transplantation Assay, Staining, Control

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Dkk3 is expressed by neurons in distinct areas of the brain . (A) Brain sections of C57BL6 mice were stained with anti-Dkk3 antibody. Brain regions were identified by morphology and location (Scale bar: 25/50 μm). (B) Respective brain sections from (A) were stained with anti-Dkk3 (red) and either anti-neuronal nuclei (NeuN, green, left panel) or anti-Glial fibrillary acidic protein (GFAP, green, right panel) antibodies. Nuclei were counterstained with DAPI (Scale bar: 15/30 μm).

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Onset and severity in the acute phase of experimental autoimmune encephalomyelitis in Dkk3-deficient mice was comparable to WT controls .

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques:

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Dkk3-deficient mice suffer from prolonged and exacerbated EAE . (A) Dkk3 −/− and WT mice were immunized s.c. with MOG 33–55 peptide in CFA. The mean EAE scores of one representative out of three experiments are shown ( n = 9). (B) The percentage of living mice with disease duration longer than 20 days was determined. Results from two independent experiments were pooled ( n = 16, * p < 0.05). (C) C57BL6 mice received either anti-Dkk3 or isotype control antibody at days 7 (1 mg/mouse), 10, 13, 16, 21 (0.5 mg/mouse) after MOG 33–55 /CFA immunization. Shown is one representative experiment out of three ( n = 10, * p < 0.05).

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Increased proportions of CD4 + CD25 + Foxp3 + regulatory T cells and IL-17-producing CD4 + T cells in the CNS but not in the draining lymph nodes (Figure H) of Dkk3 −/− at the peak of EAE . (A) EAE was induced in Dkk3 −/− and WT mice by immunization with MOG 33–55 in CFA. Mean clinical EAE score over time is displayed. At the indicated time point (black arrow, 14 days after EAE induction), half of the mice were sacrificed and lymphocytes were isolated from brain and spinal cord for analysis in (B–G) ( n = 8). (B) Percentages of CD4 + and CD8 + T cells among leukocytes in the CNS of Dkk3 −/− and WT mice. (C) Proportion of CD25 + Foxp3 + of CD4 + cells (* p < 0.05). (D–G) Isolated lymphocytes from (A) were re-stimulated in vitro with 50 μg/ml MOG 33–55 peptide for 6 h in the presence of Golgi Plug and intracellularly stained for the respective cytokines. Shown is one representative dot plot (left panel) and cumulative data of three individual experiments ( n = 8). (D) Percentages of IL-17 + of CD4 + T cells (* p < 0.05). (E) Percentages of GM-CSF + of CD4 + T cells. (F) Percentages of IFNγ + of CD4 + T cells. (G) Percentages of IFNγ + of CD8 + T cells. (H) Percentages of CD4 + and CD8 + T cells in spleen and peripheral lymph nodes (LN) of respective mice from (A) . (I) Percentages of CD25 + Foxp3 + of CD4 + cells in spleen and draining lymph nodes (LN) of respective mice from (A) . (J) Percentages of IL-17 + of CD4 + cells in spleen and draining lymph nodes (LN) of respective mice from (A) .

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Isolation, In Vitro, Staining

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Increased numbers of IFNγ-producing T cells in the CNS of EAE diseased Dkk3 −/− mice in the recovery phase . (A) EAE was induced in Dkk3 −/− and WT mice by immunization with MOG 33–55 in CFA. Mean clinical EAE score over time is displayed. At the indicated time point (black arrow, 20 days after EAE induction), mice were sacrificed and lymphocytes were isolated from brain and spinal cord for analysis in (B–G) . (B) Percentages of CD4 + and CD8 + T cells among leukocytes in the CNS of Dkk3 −/− and WT mice (* p < 0.05). (C) Proportion of CD25 + Foxp3 + of CD4 + cells. (D–G) Isolated lymphocytes from (A) were re-stimulated in vitro with 50 μg/ml MOG 33–55 peptide for 6 h in the presence of Golgi Plug and intracellularly stained for the respective cytokines. Shown is one representative dot plot (left panel) and cumulative data of three individual experiments ( n = 8). (D) Percentages of IL-17 + of CD4 + T cells. (E) Percentages of GM-CSF + of CD4 + T cells. (F) Percentages of IFNγ + of CD4 + T cells (* p < 0.05). (G) Percentages of IFNγ + of CD8 + T cells (* p < 0.05). (H) Percentages of CD4 + and CD8 + T cells in spleen and draining lymph nodes (LN) of respective mice from (A) . (I) Percentages of IFNγ + of CD4 + and CD8 + cells in spleen and draining lymph nodes (LN) of respective mice from (A) .

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Isolation, In Vitro, Staining

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Neuronal Dkk3 expression is induced by IFNγ and limits IFNγ induced gene expression in the brain . (A) Dkk3 protein levels in brain lysates of healthy and EAE-diseased WT and IFNγR1 −/− mice were measured by ELISA. EAE was induced by immunization with MOG/CFA and minimal clinical EAE score of used mice was 4. Protein amount was related to organ weight ( n = 5, ** p < 0.01). (B) Histological analysis of ex vivo cultured, primary dorsal route ganglia (DRG) stained with anti-Dkk3 (upper row) or isotype control antibody (lower row). (C) QRT-PCR data showing relative Dkk3 expression in cells from (C) ( n = 3, ** p < 0.01). (D) Dkk3 ELISA of supernatant from control and IFNγ stimulated primary DRG isolated from WT mice. DRG were stimulated for 24 h with 100 ng/ml IFNγ ( n = 3, ** p < 0.01). (E) QRT-PCR data showing relative Dkk3 expression in control and IFNγ stimulated primary DRG from IFNγR1 −/− mice. DRG were stimulated for 24 h with 100 ng/ml IFNγ ( n = 3). (F) WT and Dkk3 −/− were immunized with MOG 33–55 in CFA. Seven days later, 100 ng recombinant IFNγ in PBS was injected stereotactically into hippocampi of the respective mice. One day later, hippocampi were harvested and RNA was isolated. As controls, hippocampi of non-IFNγ-injected mice, 8 days after MOG/CFA immunization were used. Shown are qRT-PCR data of the relative expression of Gbp2 , Cxcl10 , Cxcl9 , Ccl2 (MCP-1), Ccl5 (RANTES) in respective hippocampi ( n = 8, * p < 0.05, ** p < 0.01).

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Ex Vivo, Cell Culture, Staining, Control, Quantitative RT-PCR, Isolation, Recombinant, Injection

Journal: Frontiers in Immunology

Article Title: Dickkopf-3, a Tissue-Derived Modulator of Local T-Cell Responses

doi: 10.3389/fimmu.2015.00078

Figure Lengend Snippet: Normal T-cell repertoire in Dkk3-deficient mice .

Article Snippet: Biotinylated goat anti-mouse Dkk3 antibody (R&D Systems, Minneapolis, MN, USA) at a final concentration of 1 μg/ml was used as detection antibody.

Techniques:

Journal: Nature Communications

Article Title: A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution

doi: 10.1038/s41467-024-45341-9

Figure Lengend Snippet: A Panel shows representative confocal images of immunostaining for CD68 and a wider panel of macrophage markers confirming the topographical niches of MerTK+LYVE1 high , MerTK+LYVE1 low and MerTK low CD48+ macrophage subsets in sections of comparator and frozen shoulder patient tissues. MerTK + LYVE1 high MRC1 + CD163+ and MerTK + LYVE1 low FOLR2 + ICAM1 + CD83 + FCGR3A+ subsets predominate in the capsule lining; triple positive cells are highlighted with arrow heads (Figure ). MerTK low CD48 + PTGS2 + S100A8 + IL1RA+ macrophages occupy lining and sub-lining regions. Cyan represents POPO-1 nuclear counterstain, scale bar = 20 μm. B Graph shows quantitative analysis of immunostaining for CD3 in comparator (C, n = 7 donors) and frozen shoulder (FS n = 7 donors) patient tissue sections, pooled from 3 independent experiments, statistically significant differences were calculated using a two-sided Mann-Whitney test ( P = 0.0006). Panel shows representative ChipCytometry images of T cells in sections of frozen shoulder patient tissues, residing adjacent to vascular endothelium (CD31+). Panels show staining combinations for CD4 + T cells (CD127+), CD8+ T cells (CD161 + GZMK+) and NK cells (CD56 + GZMB+), nuclei counterstained cyan/blue, scale bar = 50 μm. C Representative confocal images showing labelling for a cassette of fibroblast markers confirming the topographical niches of identified fibroblast sub-populations from Fig. in sections of comparator and frozen shoulder patient tissues. Sub-population markers include DKK3 + FMOD+, CXCL12 + PTGDS+, POSTN + CTHRC1+, ARC+CHI3L1+, MFAP5+ and CLIC5 + HBEGF + PRG4+. Cyan represents POPO-1 nuclear counterstain, scale bar = 20 μm. D Graphs show quantitative analysis (QA) of total cellularity and cellularity localised to lining and sub-lining regions. Data generated using tissue sections derived from a minimum of 7 comparator and 7 frozen shoulder donors, pooled from 3 independent experiments. Statistically significant differences were calculated using a two-sided Mann-Whitney test for lining cellularity ( P = 0.001), sub-lining cellularity ( P = 0.0003), total cellularity ( P = 0.0003). QA analysis of macrophage markers in sections of tissue biopsies collected from comparator (C) and frozen shoulder (FS) patient donors including CD68 ( n = 6 comparator and n = 6 frozen shoulder donors, P = 0.002) & CD48 ( n = 5 comparator and n = 5 frozen shoulder donors, P = 0.008). QA for markers of fibroblast activation PDPN ( P = 0.002) & CD90 ( P = 0.002), ( n = 6 comparator and n = 6 frozen shoulder donors for each marker). QA for matrix-associated markers POSTN ( P = 0.008) and CTHRC1 ( P = 0.008), ( n = 6 comparator and n = 6 frozen shoulder donors for each marker). All immunostaining data were pooled from 4 independent experiments. Statistically significant differences were calculated using two-sided Mann-Whitney tests. Bars represent median values. *** P < 0.001, ** P < 0.01.

Article Snippet: Cells were permeabilized with 0.1% Triton-X, blocked with 5% normal goat serum in PBS + 0.1% Tween-20 (PBS-T), then stained with POSTN (1:200, abcam #ab79946) and DKK3 (1:400, Proteintech #66758) overnight, followed by secondary antibodies at 1:200 (goat anti-mouse 633, goat anti-rabbit 568, Life Technologies).

Techniques: Immunostaining, MANN-WHITNEY, Staining, Generated, Derivative Assay, Activation Assay, Marker

Journal: Nature Communications

Article Title: A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution

doi: 10.1038/s41467-024-45341-9

Figure Lengend Snippet: A Heatmap showing selected genes that showed significant variance in expression between capsular fibroblasts from frozen shoulder patients co-incubated with MerTK high , MerTK low MDMs or fibroblasts in isolation (DESeq2, LRT test, BH adjusted P < 0.05). See also Supplementary Fig. . B Dot plot shows GO biological processes over-represented in the sets of genes down-regulated in capsular fibroblasts by incubation with MerTK high MDMs (group 1) or up-regulated by incubation with MerTK high MDMs (group 2). C The boxplots show the predicted proportions of fibroblast subsets identified in the single-cell analysis (Fig. ) present in the untreated, MerTK high -MDM co-cultured and MerTK low -MDM co-cultured fibroblasts ( n = 3 frozen shoulder patient donors in 2 independent experiments, bars show median value (median; centre, box limits; lower and upper quartile, whiskers; 1.5 IQR), deconvolution performed with MuSiC). D Selected predicted ligand-receptor interactions between MerTK+LYVE1+ myeloid and DKK3 + FMOD+ or POSTN + ACAN+ fibroblast sub-populations generated from differentially expressed genes from comparator (n = 6 donors) and frozen shoulder ( n = 4 donors), sub-populations as in Fig. (NATMI analysis). E The scatter plot shows the expression change of predicted ligand-receptor interactions between MerTK+LYVE1+ myeloid and DKK3 + FMOD+ or POSTN + ACAN+ fibroblast sub-populations in frozen shoulder relative to comparator patient tissues. F Protein-protein network association analysis of receptors (blue) highly expressed in frozen shoulder patient fibroblasts identified a candidate interaction with RUNX2 (analysis performed with IntAct). G Single-cell PySCENIC gene regulatory network analysis of fibroblasts (Fig. , all clusters) identified a connection between the expression of RUNX2 and matrix associated genes including CDH11, MMP14, MMP13 and SPP1 (pink) in POSTN + ACAN+ fibroblasts.

Article Snippet: Cells were permeabilized with 0.1% Triton-X, blocked with 5% normal goat serum in PBS + 0.1% Tween-20 (PBS-T), then stained with POSTN (1:200, abcam #ab79946) and DKK3 (1:400, Proteintech #66758) overnight, followed by secondary antibodies at 1:200 (goat anti-mouse 633, goat anti-rabbit 568, Life Technologies).

Techniques: Expressing, Incubation, Isolation, Single-cell Analysis, Cell Culture, Generated

Journal: Nature Communications

Article Title: A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution

doi: 10.1038/s41467-024-45341-9

Figure Lengend Snippet: scRNAseq was performed on 12, 15 and 17 pcw developing human shoulder joint tissues. A UMAP shows the major cell types comprising the developing foetal shoulder joint (resolution = 0.9). B UMAP (resolution = 0.6) shows identified myeloid populations in the developing shoulder joint (12, 15 and 17 pcw). Sub-clustering of myeloid populations revealed 7 distinct clusters including MERTK + LYVE1+ and MERTK+TIMD4+ populations. C The plot shows the same foetal cell UMAP with labels transferred from the adult shoulder capsule myeloid cells (Fig. ) (scArches analysis). D UMAP (resolution = 0.7) showing CD45–COL1A1+ stromal cell clusters in the developing shoulder joint (12, 15 and 17 pcw). E The plot shows the same UMAP of developing shoulder stromal cells with labels transferred from the adult clusters (Fig. ) (scArches analysis). F The Sankey plot shows the mapping between the clusters identified in the developing shoulder stromal cells (D) (left) and the transferred labels from the adult fibroblast populations (right). G – J Representative Cell DIVE and respective H&E stained images of histological sections of foetal shoulder joint at 17 post conception weeks (pcw) development stage. G , H Sections stained for a panel of markers to identify fibroblasts in the shoulder capsule during development (FMOD, DKK3, POSTN, CLU). DAPI nuclear counterstain is dark blue, scale bar = 20 μm. I High magnification image showing the cell types identified in foetal shoulder capsule including macrophages (CD68, CD163, MerTK) and fibroblasts (PDPN, CLU, AXL, GAS6) in the capsule lining region at 17 pcw. Scale bar = 20 μm. J High magnification image of the foetal shoulder capsule (17 pcw) showing immunostaining for macrophage markers MerTK, LYVE1, CD206 and CD163 in the capsule lining region. DAPI nuclear counterstain is dark blue, white arrows mark MerTK+ macrophages. Scale bar = 20 μm.

Article Snippet: Cells were permeabilized with 0.1% Triton-X, blocked with 5% normal goat serum in PBS + 0.1% Tween-20 (PBS-T), then stained with POSTN (1:200, abcam #ab79946) and DKK3 (1:400, Proteintech #66758) overnight, followed by secondary antibodies at 1:200 (goat anti-mouse 633, goat anti-rabbit 568, Life Technologies).

Techniques: Staining, Immunostaining