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: 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

Journal: eLife

Article Title: Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

doi: 10.7554/eLife.41460

Figure Lengend Snippet: ( A ) Serum-starved K562 cells were treated with PBS (control) or Wnt3a (100 ng/ml) or Wnt3a + Dickkopf peptide (DKK1, 200 ng/ml) for 24 hr prior to EV isolation. Biochemical analysis of cellular (Cells) and EV fractions was performed by immunoblotting of extracts using antibodies against antigens shown. Detection of ß-Catenin was carried out using cytosolic fractions. ( B ) Quantitation of EV marker protein levels from EV immunoblots shown in ( A ). ( C ), Serum-starved K562 cells were treated with DMSO (control) or CHIR99021 (10 µM) for 24 hr prior to EV isolation. Biochemical analysis of EV fractions was performed as in ( A ). Representative immunoblots for a control experiment along with two replicate CHIR99021-treated samples are shown. ( D ) Quantitation of protein levels from EV immunoblots in ( C ). ( E ) Quantitation of LAMP1 protein from cellular fractions in ( C ). Molecular mass marker mobility is shown at the left of immunoblot panels in kilodaltons. t test: *p<0.05; **p<0.01; error bars represent SEM; n ≥ 3. ( F ) Flow cytometry determination of LAMP1 levels in anti-LAMP antibody-labeled control and CHIR99021-treated cells. Confocal images at right show lysosomes stained with anti-LAMP1 antibody (green) and nuclei labeled with DAPI (blue). Bar,10 µm.

Article Snippet: The next day, the medium was replaced with fresh EV-free RPMI supplemented with 100 ng/ml recombinant Wnt3a protein (R and D Systems), or Wnt3a combined with 200 ng/ml recombinant DKK1 protein (R and D Systems) and EVs were collected 24 hr later.

Techniques: Control, Isolation, Western Blot, Quantitation Assay, Marker, Flow Cytometry, Labeling, Staining

Journal: Frontiers in Microbiology

Article Title: Core Fucosylation of Intestinal Epithelial Cells Protects Against Salmonella Typhi Infection via Up-Regulating the Biological Antagonism of Intestinal Microbiota

doi: 10.3389/fmicb.2020.01097

Figure Lengend Snippet: Wnt signaling pathway mediates the core fucosylation of Caco-2 by S. Typhi infection in vitro . (A) The expression of 7-Frz, β-catenin and core fucosylation of Caco-2 cells were up-regulated post infection. Caco-2 cells were incubated with S. Typhi for 30 min, washed, and incubated in fresh DMEM with 10% fetal bovine serum for 30, 60, or 120 min. Control group indicates without bacterial treatment. Data are shown as mean values ± SEM (Control, n = 3; 30 min, n = 3; 60 min, n = 3; 120 min, n = 3; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001). (B) The expression of 7-Frz and β-catenin of Caco-2 cells was up-regulated after application of Dkk-1. The cells were divided into control group (group Control) and experimental group (group 10, 100, and 200 ng) according to the completely random method. The dose of Dkk-1 for experimental group: 10, 100, and 200 ng. Data are shown as mean values ± SEM (Control, n = 3; 10 ng, n = 3; 100 ng, n = 3; 200 ng, n = 3; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001). (C) Caco-2 cells were classified into Control group (cells without any treatment), Dkk group (cells were treated by 2 × 10 4 ng/mL, 10 μl Dkk-1 for 48 h), S. Typhi group (cell were incubated with S. Typhi strain for 30 min, and incubated with medium for 60 min), S. Typhi + Dkk group (cell were incubated with S. Typhi strain for 60 min, and incubated with medium for 60 min then treated by 2 × 10 4 ng/mL, 10 μl Dkk-1 for 48 h). Data are shown as mean values ± SEM (Control, n = 3; Dkk-1, n = 3; S. Typhi, n = 3; S. Typhi + Dkk-1, n = 3; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001). (D) The interaction between β-catenin/TCF complexes and Fut8 promoter was analyzed by ChIP analysis. TCF can directly bound to the Fut8 promoter, and S. Typhi infection increased the inputs. Caco-2 cells were incubated with S. Typhi for 30 min, washed, and incubated in fresh DMEM with 10% fetal bovine serum for 60 min. Control group indicates without bacterial treatment. Normalized inputs of chromatin DNA from Caco-2 cells were pulled down with anti-β-catenin or negative IgG antibodies. Data are shown as mean values ± SEM (Control, n = 3; S. Typhi, n = 3; * p < 0.05).

Article Snippet: The group 10 ng was given 10 3 ng/mL Dkk-1 (HY-P7155A, MCE) 10 μl, the group 100 ng was given 10 4 ng/mL Dkk-1 10 μl, the group 200 ng was given 2 × 10 4 ng/mL Dkk-1 10 μl, and the same volume PBS was added into group 0.

Techniques: Infection, In Vitro, Expressing, Incubation, Control