Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: (A) Real-time PCR showing relative mRNA expression of DKK4 and β-catenin (CTNNB1) in 46 bulk human GIST tumors. Lines represent the medians. Prim/UT– untreated primary GIST (n = 22), Met/Res – resistant metastatic GIST (n = 24). Student’s t test; ***P < 0.001. (B) Representative (left) and quantitative (right) staining of β-catenin in a human GIST tissue microarray of surgical specimens from the time period 1982–2004. The percentage of relative intensity of β-catenin versus total area was calculated using Metamorph software. Medians are indicated. Student’s t test; ***P < 0.001. Prim/UT– primary, untreated GIST; Met/UT– metastatic, untreated GIST; Met/Res – metastatic, resistant GIST. (C) Representative staining of β-catenin in 46 additional human GIST specimens. Scale bar, 20 μm. (D) The corresponding β-catenin intensity was scored as negative to weak (0), moderate (1+), or high (2+). The fraction of high β-catenin intensity score is significantly higher in metastatic, resistant GIST (54.5%) than in primary, untreated GIST (12.5%). (E) Western blot analysis of nuclear active β-catenin isolated from high β-catenin-expressing human GISTs. (F) Relative mRNA expression of FDZ6, FDZ7, and LRP6 in human GIST cell lines and freshly isolated KIT+ tumor cells from 8 human specimens as detected by real-time PCR, compared to Caco-2 colorectal cancer cells. Prim/UT– untreated primary GIST, Met/Res – resistant metastatic GIST, Met/Sen – sensitive metastatic GIST.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Microarray, Software, Western Blot, Isolation

Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: (A) Immunoblots of GIST T1, GIST882, and murine S2 whole cell lysates after treatment with either 150 ng/ml Wnt3a or control PBS for 4h. (B) Real-time PCR of Axin 2 and CCND1 (cyclin D1) mRNA expression after treatment with either 150 ng/ml Wnt3a or control PBS for 4h. Bars indicate means ± SEM. Student’s t test; *P < 0.05. (C) TCF/LEF reporter activity in GIST T1 cells in the presence of Wnt3a and/or β-catenin overexpression. GIST T1 cells were co-transfected with a negative reporter or TCF/LEF reporter and a mock or β-catenin expression plasmid. 48h after transfection, cells were treated with either 150 ng/ml of Wnt3a or control PBS for 4h. Relative luciferase signaling (indicating TCF/LEF activity) was determined after normalizing to renilla luciferase (indicating transfection efficiency), and expressed as relative fold changes compared to indicated control. All bars, means ± SEM. Student’s t test; *P < 0.001. (D) Immunoblots of whole-cell lysates of GIST T1 cells that had been transfected with mock or DKK4 expression plasmid and 48h later treated with either 150 ng/ml of Wnt3a or PBS for 4h. Western blot bands were quantified by Image J Software and normalized to GAPDH, and then expressed as the fold-change compared to pCMV6-mock control. (E) Cell viability assay (Dojindo) of human GIST T1 cells following 72h of transfection of control siRNA or b-catenin smartpoolsiRNA. Bars, mean ± SEM. Student’s t test; *P < 0.05.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Over Expression, Transfection, Plasmid Preparation, Luciferase, Software, Viability Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 3. Upregulation of Dkk1 expression in pagetic osteoblasts and BMSCs. (A) The relative expression of Dkk1 was determined by real-time PCR, and values are expressed relative to the mean level of expression in the nonpagetic groups, which have been normal- ized to 1. The number of samples was the same as in Fig. 2. (B) The concentration of secreted Dkk1 protein in conditioned media samples as determined by ELISA. The con- ditioned media samples were collected dur- ing the last 72 h of incubation from the cell cultures described above. Data in A and B are presented as mean ± SE. ap < 0.05 and bp < 0.01 vs. control. (C) Correlation be- tween levels of Dkk1 mRNA in cells and se- creted Dkk1 protein in conditioned media. Levels of Dkk1 mRNA are expressed rela- tive to the expression of ribosomal RNA. The correlation was calculated using Deming (model II) linear regression (r 0.88, p < 0.0001).

Article Snippet: A total of 100 l of standard or sample was loaded per well and incubated overnight at 4°C, washed, and incubated with biotinylated goat antihuman Dkk1 IgG (R&D Systems) diluted to a concentration of 0.2 g/ml in dilution buffer.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 7. Schema showing possible effects and interactions of the changes in osteoblast gene expression shown in this study. Solid lines denote production of a factor by that cell, and broken lines indicate a regulatory influence of a factor. The overproduction of IL-1 and IL-6 by the pagetic osteoblast will result in osteoclast proliferation, leading to further increases in IL-6 levels in the bone marrow microenvironment. Increased pro- duction of Dkk1 by the pagetic osteoblast will further increase IL-6 levels and reduce osteoblast proliferation. This combination of effects could account for the development of lytic lesions in early phase Paget’s disease. Over time, both the excess of Dkk1 and of IL-6 will result in increased differentiation of osteoblasts, thus promoting mineralization. This could contribute to the development of sclerosis in longer-standing pagetic lesions.

Article Snippet: A total of 100 l of standard or sample was loaded per well and incubated overnight at 4°C, washed, and incubated with biotinylated goat antihuman Dkk1 IgG (R&D Systems) diluted to a concentration of 0.2 g/ml in dilution buffer.

Techniques: Gene Expression

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 3. Upregulation of Dkk1 expression in pagetic osteoblasts and BMSCs. (A) The relative expression of Dkk1 was determined by real-time PCR, and values are expressed relative to the mean level of expression in the nonpagetic groups, which have been normal- ized to 1. The number of samples was the same as in Fig. 2. (B) The concentration of secreted Dkk1 protein in conditioned media samples as determined by ELISA. The con- ditioned media samples were collected dur- ing the last 72 h of incubation from the cell cultures described above. Data in A and B are presented as mean ± SE. ap < 0.05 and bp < 0.01 vs. control. (C) Correlation be- tween levels of Dkk1 mRNA in cells and se- creted Dkk1 protein in conditioned media. Levels of Dkk1 mRNA are expressed rela- tive to the expression of ribosomal RNA. The correlation was calculated using Deming (model II) linear regression (r 0.88, p < 0.0001).

Article Snippet: Microtiter plates (Greiner) were coated with 100 l of anti-Dkk1 polyclonal antibody (R&D Systems) at a concentration of 1 g/ml in PBS, pH 7.2.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 7. Schema showing possible effects and interactions of the changes in osteoblast gene expression shown in this study. Solid lines denote production of a factor by that cell, and broken lines indicate a regulatory influence of a factor. The overproduction of IL-1 and IL-6 by the pagetic osteoblast will result in osteoclast proliferation, leading to further increases in IL-6 levels in the bone marrow microenvironment. Increased pro- duction of Dkk1 by the pagetic osteoblast will further increase IL-6 levels and reduce osteoblast proliferation. This combination of effects could account for the development of lytic lesions in early phase Paget’s disease. Over time, both the excess of Dkk1 and of IL-6 will result in increased differentiation of osteoblasts, thus promoting mineralization. This could contribute to the development of sclerosis in longer-standing pagetic lesions.

Article Snippet: Microtiter plates (Greiner) were coated with 100 l of anti-Dkk1 polyclonal antibody (R&D Systems) at a concentration of 1 g/ml in PBS, pH 7.2.

Techniques: Gene Expression

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 1. In vitro evaluation of the proper effect of the BHQ880 antibody over Dkk1 protein neutralization. (A) Representative images of B1a/7TGC cell reporter line activation when stimulated by the GSK-3 inhibitor CHIR99021 (positive control) or by increasing doses of recombinant mouse Wnt3a (mWnt3a), and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. Non-treated (NT); # vs. 0 ng/mL mWnt3a. (B) Representative images of B1a/7TGC cell reporter line inactivation when previously stimulated cells with 150 ng/mL of mWnt3a were turned off through the addition of increasing doses of recombinant rDkk1, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; # vs. 0 ng/mL rDkk1. (C) Representative images of the BHQ880 neutralizing effect over rDkk1 protein in the B1a/7TGC reporter cells stimulated with 150 ng/mL of mWnt3a, together with 250 ng/mL of rDkk1 and increasing doses of BHQ880 antibody, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; & vs. Wnt3a; # vs 0 ng/mL BHQ880. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between groups. In all cases, scale bars = 50 μm and the data are presented as the mean + SEM. **p <0.01; ***p <0.001; #p <0.05; ##p <0.01; ###p <0.001; &&p <0.01.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: In Vitro, Neutralization, Activation Assay, Positive Control, Recombinant

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 2. Protein expression of Dkk1 in the healthy and injured spinal cord. The figure shows representative images from the immunohistochemical evaluation of Dickkopf-1 (Dkk1) protein expression in the non-lesioned (NL) (n = 5) spinal cord (A) and after SCI at 24 hours post-injury (hpi) (n = 4), and 3 days post-injury (dpi), 7 dpi (n = 4), 14 dpi (n = 5), 28 dpi (n = 3), and 42 dpi (n = 5) at the lesion epicenter (B-G). Dashed line squares in A-G indicate the areas shown in the corresponding higher magnification images. Scale bars = 500 μm. Inserts scale bars = 50 μm. (H) Densitometric analysis of Dkk1 immunolabeling in the NL spinal cord and at the injury epicenter after SCI from 24 hpi to 42 dpi. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between NL and the different times post-injury. In all cases, the data are presented as the mean + SEM. **p <0.01; ***p <0.001.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Expressing, Immunohistochemical staining, Immunolabeling

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 3. Analysis of the circulating levels of Dkk1 in Serum samples from non- lesioned (NL) and lesioned rats at 24 hours after damage (SCI). The figure shows data obtained from the quantitative analysis of serum circulating Dkk1 protein levels measured by ELISA in NL animals (n = 9) and injured animals 24 hours after SCI (n = 9). No significant differences were observed between groups. A two-tailed t test was used to determine the existence of significant differences between groups. The data represent the mean ± SEM.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test