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: The Journal of Biological Chemistry

Article Title: Calreticulin regulates a switch between osteoblast and chondrocyte lineages derived from murine embryonic stem cells

doi: 10.1074/jbc.RA119.011029

Figure Lengend Snippet: Recombinant DKK1 increases chondrocyte specification while blocking osteoblast specification. Differentiated WT ESCs were stained with either Alcian blue or von Kossa as indicated ( A ), treated without or with rbDKK1 for days 6–17 of osteoblast differentiation. B , RT-qPCR analysis of chondrocyte or osteoblast markers in WT ESCs differentiated in the absence (■) or presence (▿) of rbDKK1 for days 6–17. C , Western blot analysis of whole-cell lysates harvested at the indicated times from ESCs during osteoblast differentiation in the absence (■, WT ESCs; ♦, crt −/− ESCs) or presence of mouse rbDKK1 for days 6–17 (▿, WT ESCs; ♢, crt −/− ESCs). Utilized antibodies were specific to SOX9 and GAPDH as an internal control. The graph is a quantitative representation of the density of the Western blotting bands of SOX9 normalized to internal control GAPDH. Data are expressed as means ± S.D. ( error bars ), n ≥ 3; two-way ANOVA of transcript levels ( B ) and band densities ( C ). B , Sox9 , p < 0.0001, F = 31.25; aggrecan, p < 0.0001, F = 94.4; osterix, p = 0.0073, F = 7.874; osteocalcin, p = 0.0007, F = 13.24. C , p < 0.0001 and F = 24.55 for WT control and crt −/− ESCs data set; p < 0.0001 and F = 48.83 for WT control ESCs treated or not with rbDKK1; p = 0.3412 and F = 0.9386 for crt −/− ESCs treated or not with rbDKK1. Bonferroni post hoc test was as indicated: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ψ, p < 0.001 for WT control ESCs treated or not with rbDKK1; ***, p < 0.001 for WT control and crt −/− ESCs ( C ). D , confocal images of immunolocalization using SOX9, type II collagen, or osterix antibodies on WT ESCs at day 19 of the osteogenic differentiation protocol in the presence of rbDKK1 on days 6–17. Dual-channel grayscale images of a single field are displayed with the labeled protein of interest in the left panel and DAPI-stained nuclei in the right panel , and the RGB panel is a merged image of green (protein of interest) and blue (DAPI). See E for the WT ESC nontreated control. Scale bar , 50 μm.

Article Snippet: Differentiating cells were treated with the inhibitor of intranuclear transport of NFAT A-285222 ( M r 416.28) ( ) (a generous gift from AbbVie) at 5 μg/ml, a 3 μ m concentration of the GSK inhibitor CHIR99021 (Tocris), or 100 ng/ml mouse recombinant DKK1 (R&D Systems), each continuously for days 6–17.

Techniques: Recombinant, Blocking Assay, Staining, Quantitative RT-PCR, Western Blot, Control, Labeling

Journal: Cell Regeneration

Article Title: RhoA/Rock activation represents a new mechanism for inactivating Wnt/β-catenin signaling in the aging-associated bone loss

doi: 10.1186/s13619-020-00071-3

Figure Lengend Snippet: RhoA/Rock constraints Wnt/β-catenin signaling and osteoblastic differentiation. a-c RhoA activation assays in primary murine calvarial osteoblasts (PMCOBs) stimulated with rWnt3a at 100 ng/ml or the indicated concentrations for the indicated times or 60 min in the presence or absence of recombinant Dkk1 (rDkk1) at 100 ng/ml. d , e Western analyses of β-catenin in cytosolic and nuclear fractions of PMCOBs with the indicated genotypes of Col1-Cre ( Cre ), Col1-Cre;caRhoA +/− ( Cre;caRhoA +/− ) or Col1-Cre;dnRhoA +/− ( Cre;dnRhoA +/− ), and in the presence or absence of rWnt3a for 3 h. f Western analyses of β-catenin (β-cat) in cytosolic and nuclear fractions of PMCOBs treated with or without Fasudil at 20 μM and stimulated with or without rWnt3a for 3 h. g-i Alp activity and mineralization nodule formation assays and their quantification in PMCOBs with the indicated genotypes and stimulated with or without rWnt3a at 100 ng/ml for 48 h and 21 d, respectively. Mean ± SEM, * p < 0.05, **,++ p < 0.01, n = 4, Tukey-Kramer multiple comparisons test

Article Snippet: Recombinant mouse active Wnt3a (rWnt3a), Wnt5a (rWnt5a), and Dkk1 (rDkk1) were from StemRD (Burlingame, CA).

Techniques: Activation Assay, Recombinant, Western Blot, Activity Assay

Journal: Cell Regeneration

Article Title: RhoA/Rock activation represents a new mechanism for inactivating Wnt/β-catenin signaling in the aging-associated bone loss

doi: 10.1186/s13619-020-00071-3

Figure Lengend Snippet: RhoA interacts genetically with Dkk1 in the limb bud ectoderm of mouse embryos. a Representative immunostaining for p-Rock2 in the E10.5 limb buds with the indicated genotypes. White dot lines separate the apical ectodermal ridge (AER) from the zone of polarizing activity (ZPA). b-d Skeletons and/or limbs of E16.5 embryos with the indicated genotypes. e , f Western analyses in PMCOBs with the indicated genotypes and stimulated with or without rWnt3a at 100 ng/ml for 1 h in the presence or absence of rDkk1 at 100 ng/ml. Phosphorylated proteins were normalized to their total amounts, respectively. g , h H&E and TUNEL staining in E10.5 forelimb bud sections ( g ) and whole-mount in situ hybridization of E10.5 forelimb buds ( h ). Dot lines separate the AER from the ZPA. Ventral view for all limb buds, anterior to the lower and posterior to the upper. FL: forelimb, HL: hindlimb

Article Snippet: Recombinant mouse active Wnt3a (rWnt3a), Wnt5a (rWnt5a), and Dkk1 (rDkk1) were from StemRD (Burlingame, CA).

Techniques: Immunostaining, Activity Assay, Western Blot, TUNEL Assay, Staining, In Situ Hybridization

Journal: Cell Regeneration

Article Title: RhoA/Rock activation represents a new mechanism for inactivating Wnt/β-catenin signaling in the aging-associated bone loss

doi: 10.1186/s13619-020-00071-3

Figure Lengend Snippet: FZD receptors and Dkk1 coordinate RhoA/Rock activation to destabilize β-catenin in the BMMSCs from elderly mice. a-d Quantitative RT-PCR analyses for the indicated mRNA levels of BMMSCs isolated form 2- or 8-month-old mice and treated with or without rWnt3a at 100 ng/ml for 48 h. Mean ± SD, ** p < 0.01, n = 6, Tukey-Kramer multiple comparisons test. e-g Western blotting analyses in the BMMSCs isolated form 2- or 8-month-old mice and treated with or without rWnt3a/rWnt5a at 100 ng/ml in the presence or absence of rDkk1 at 100 ng/ml for 3 h. h An integrated working model of β-catenin signaling mediated by RhoA/Rock in the regulation of aging-associated bone loss. In the BMMSCs from young subjects, APC/Axin/Gsk3β signaling mediated by FZD1, FZD4, and FZD7 overwhelms the RhoA/Rock/Jak/Gsk3β signaling mediated by FZD3, FZD6, and FZD8 to stabilize β-catenin and, in turn, enhance bone formation. However, in the BMMSCs from elderly subjects, the activation of RhoA/Rock/Jak/Gsk3β signaling mediated by FZD3, FZD6, and FZD8, in combination with the inactivation of APC/Axin/Gsk3β signaling mediated by Dkk1, Sost, and FZD1, FZD4, and FZD7 results in the destabilization of β-catenin and the subsequent attenuation of bone formation

Article Snippet: Recombinant mouse active Wnt3a (rWnt3a), Wnt5a (rWnt5a), and Dkk1 (rDkk1) were from StemRD (Burlingame, CA).

Techniques: Activation Assay, Quantitative RT-PCR, Isolation, Western Blot