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: PloS one

Article Title: Wnt pathway activity in breast cancer sub-types and stem-like cells.

doi: 10.1371/journal.pone.0067811

Figure Lengend Snippet: Figure 3. Gene expression analysis of Wnt signalling in monolayer (Mono) and anoikis resistant (AR) cells of normal breast cell lines (N), ER-ve and ER+ve breast cancer cell lines. A) Protein expression of activated B-catenin (unphosphorylated), Lef1, Axin2, DKK1 and B-actin (housekeeper) in MCF7 monolayer and AR cells. B) Cluster analysis was performed using the fold change in expression

Article Snippet: MCF10a, MCF7, MDA-MB-231, primary human normal breast cells and primary human invasive breast cancer cells were plated into MS culture and treated with a single dose of human recombinant DKK1 (R and D systems) at increasing concentrations (0–100ng/ml).

Techniques: Gene Expression, Expressing

Journal: PloS one

Article Title: Wnt pathway activity in breast cancer sub-types and stem-like cells.

doi: 10.1371/journal.pone.0067811

Figure Lengend Snippet: Figure 4. Modulation of Wnt signalling in normal and breast cancer cell lines. Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) MCF10a cells (Wnt3a) B) MCF7 cells (Wnt3a) C) MDA-MB-231 cells (Wnt3a) D) MCF10a cells (DKK1) E) MCF7 cells (DKK1) F) MDA-MB-231 cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * ,0.01 ** ,0.001 generated by a T-test. G) Image of a MCF10a mammosphere H) Image of an MCF7 mammosphere I) Image of an MDA-MB-231 mammosphere. Scale bar represents 50 mM. doi:10.1371/journal.pone.0067811.g004

Article Snippet: MCF10a, MCF7, MDA-MB-231, primary human normal breast cells and primary human invasive breast cancer cells were plated into MS culture and treated with a single dose of human recombinant DKK1 (R and D systems) at increasing concentrations (0–100ng/ml).

Techniques: Cell Culture, Control, Generated

Journal: PloS one

Article Title: Wnt pathway activity in breast cancer sub-types and stem-like cells.

doi: 10.1371/journal.pone.0067811

Figure Lengend Snippet: Figure 5. Modulation of Wnt signalling in normal and primary breast cancer samples (Normal n = 3; ER+ve n = 3; ER-ve n = 3). Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) primary normal breast cells (Wnt3a) B) ER+ve primary breast cancer cells (Wnt3a) C) ER-ve primary breast cancer cells (Wnt3a) D) primary normal breast cells (DKK1) E) ER+ve primary breast cancer cells (DKK1) F) ER2ve primary breast cancer cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * .0.01 ** .0.001 generated by a T-test. G) Image of a normal primary mammosphere H) Image of an ER positive primary tumour mammosphere I) Image of an ER negative primary tumour mammosphere. Scale bar represents 50 mM. doi:10.1371/journal.pone.0067811.g005

Article Snippet: MCF10a, MCF7, MDA-MB-231, primary human normal breast cells and primary human invasive breast cancer cells were plated into MS culture and treated with a single dose of human recombinant DKK1 (R and D systems) at increasing concentrations (0–100ng/ml).

Techniques: Cell Culture, Control, Generated

Journal: Oncotarget

Article Title: Positive feedback loop of hepatoma-derived growth factor and β-catenin promotes carcinogenesis of colorectal cancer

doi:

Figure Lengend Snippet: A. - B. β-catenin knockdown significantly suppressed HDGF mRNA expression in HCT116 A. and HT29 B. cells by real-time PCR analysis, respectively; C. - E. β-catenin knockdown inhibited HDGF protein expression in HCT116 cells C. and mainly inhibited nuclear HDGF, β-catenin, c-Myc, cyclin D1, MMP9 and phos-GSK-3β (Ser9) protein expression in HCT116 D. and HT29 E. cells; F. Recombinant Wnt3a and DKK1 increased and decreased HDGF and β-catenin expression in HCT116 cells, respectively; G. - I. Recombinant Wnt3a and DKK1 increased and decreased nuclear and cytoplasmic HDGF, β-catenin, c-Myc, cyclin D1, MMP9 and phos-GSK-3β (Ser9) protein expression in HCT116 G. , I. and LOVO H. cells by Western blot analysis, respectively.

Article Snippet: To further verify the effect of β-catenin on HDGF expression in CRC cells, HDGF and β-catenin protein expressions in HCT116 were induced by 100ng/ml human recombinant Wnt3a (R&D SYSTEMS) and inhibited by 200ng/ml human recombinant DKK1 (R&D SYSTEMS) for 48 hours by Western blot analysis, respectively (Figure ).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Recombinant, Western Blot