dkk1 (rdkk1)  (StemRD Inc)

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: S100A4-induced expression alterations of DKK1 in CRC cell lines HCT116, HAB92, and HAB68. ( a ) Increased S100A4 expression and decreased DKK1 expression in HAB92/S100A4 cells vs. HAB92 cells (part 1) and HAB92/S100A4 cells vs. HAB92/vector cells (part 2). ( b ) Overexpression of S100A4 in HAB92/S100A4 cells on mRNA and protein level. Down-regulation of DKK1 mRNA expression ( c ) and of extracellular DKK1 ( d ) in HAB92/S100A4 cells. ( e ) S100A4 expression in HCT116, HAB68, and HAB92 cells on mRNA and protein levels; lowest S100A4 expression in HAB92 cells. Differential expression of DKK1 in HCT116, HAB68, and HAB92 cells on mRNA ( f ) and extracellular protein ( g ) levels; highest expression in HAB92 cells.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Plasmid Preparation, Over Expression, Quantitative Proteomics

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Inverse expression correlation of S100A4 and DKK1 in a panel of 13 CRC cell lines. ( a ) Relative mRNA expression level of S100A4 and DKK1 determined by gene-specific qRT PCR. ( b ) Western blot analysis of S100A4 expression. GAPDH served as loading control. ( c ) ELISA of extracellular amounts of human DKK1 in culture medium in the fold of HCT116. Names in bold indicate mutated β-catenin: HCT116—S45Δ, SW48—S33Y, LIM1215—T41A, LS513—A5-80Δ, LS174T—S45F. * indicates wt for both APC and β-catenin .

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Expression regulation of DKK1 in CRC cells involves the transcription factor ATF5 and S100A4 but on different sites of the promoter. ( a ) ChIP assays of gene-specific pull-downs in HAB68 and HAB92 cells confirm the binding of ATF5 to the DKK1 promoter. Unspecific immunoglobulin served as the negative control and RNA polymerase II as an indicator of general DKK1 transcription. ( b ) Ectopic ATF5 expression in HAB68 cells increased DKK1 promoter-driven luciferase activity, while ectopic expression of S100A resulted in decreased reporter signal. DKK1 promoter fragments: −2238 bp–+112 bp (2.35 kb); −231 bp–+112 bp (0.34 kb).

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Binding Assay, Negative Control, Luciferase, Activity Assay

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Transcriptional cross-regulation of DKK1 and S100A4 affects cellular motility. Relative DKK1 mRNA expression ( a ) and DKK1 protein secretion ( b ) in HAB92/shDKK1 cells. ( c ) Increase of S100A4 mRNA and protein expression in HAB92/shDKK1 cells. ( d ) Relative S100A4 mRNA and protein expression in HCT116/shS100A4 cells. ( e ) Increase of relative DKK1 mRNA expression in HCT116/shS100A4 cells. ( f ) Decrease of S100A4 mRNA expression level following treatment with rDKK1. ( g ) Decrease of cellular motility by rDKK1 treatment in HCT/vector cells is rescued by ectopic S100A4 expression in HCT116/S100A4 cells.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Plasmid Preparation

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: S100A4 reduction restores endogenous DKK1 expression in vivo. Relative mRNA expression of S100A4 ( a ) and DKK1 ( b ) in intrasplenic tumor tissue of xenografted mice receiving the systemic application of S100A4-specific shRNA expression plasmids. Immunostaining of S100A4 ( c ), DKK1 ( d ), and background control ( e ) of two independent samples per group of intrasplenic xenograft tumor tissue. Images were taken at 20× and 40× magnification, and scale bars represent 200 µm and 100 µm, respectively. Quantification of protein-specific immunostaining confirms the cross-regulation of S100A4 ( f ) and DKK1 ( g ) in vivo. Quantified expression of target genes occurred in triplicates of eight independent animal tumors.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, In Vivo, shRNA, Immunostaining, Control

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Correlation analysis of S100A and DKK1 mRNA expression of combined GEO datasets of CRC microarray analyses. Expression levels of target genes were normalized to G6PDH.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Microarray

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Combination of S100A4 and DKK1 for improved prognosis of OS CRC patients. DKK1, as well as S100A4, mRNA expression levels were determined by qRT PCR in micro-dissected tumor cell populations of primary tumors of stages I–IV ( n = 41). The cut-off values to distinguish low and high expression levels were determined by ROC analyses. ( a ) OS of CRC patients, based on the S100A4 mRNA expression in the primary tumor. ( b ) OS of CRC patients, based on the DKK1 mRNA expression in the primary tumor. ( c ) OS of CRC patients, based on the combination of S100A4 and DKK1 expression in the tumor. Cut-off values for the respective gene and analysis are indicated by the axis labels.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Combination of S100A4 and DKK1 for improved prognosis of OS and MFS of CRC patients. DKK1 and S100A4 mRNA expression levels were determined by qRT PCR in micro-dissected tumor cell populations of primary, not yet metastasized, tumors of stages I, II, and III ( n = 60). The cut-off values to distinguish low and high expression levels were determined by ROC analyses (highest Youden index: S100A4—2.68; DKK1—0.21). Survival analysis was performed with the Kaplan–Meier estimator, with a chi-square multiple comparison. OS ( a ) and MFS ( b ) of CRC patients, based on the S100A4 mRNA expression in the primary tumor. OS ( c ) and MFS ( d ) of CRC patients, based on the DKK1 mRNA expression in the primary tumor. OS ( e ) and MFS ( f ) of CRC patients, based on the combination of S100A4 and DKK1 expression in the tumor.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR, Comparison

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Expression regulation of DKK1 in HAB68 and HAB92 cells occurs near the transcription start site. ( a ) Graphical representation of the human DKK1 promoter. Predicted binding sites of transcription factor complexes containing TCF and CREB-family members are indicated in black and grey, respectively. ( b ) Relative DKK1 promoter-driven luciferase activity in HAB68 cells is significantly lower for all DKK1 promoter constructs compared to HAB92 cells. * statistically significant.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Construct

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: Induced ectopic expression of S100A4 in xenograft tumors results in a reduced abundance of human DKK1 in mouse plasma. ( a ) Schematic representation of the lentiviral vector for doxycycline-induced S100A4 expression. The respective control vector was generated without the coding sequence for S100A4 (not shown). Either vector was lentivirally transduced into HAB92 cells. ( b ) Plasma samples of mice intrasplenically transplanted with HAB92/tetON-S100A4 or HAB92/tetON-ctrl cells were taken after 15 days with or without doxycycline treatment. Immunostaining with human-specific DKK1 antibodies after WB showed reduced hDKK1 abundance in plasma samples of mice with induced S100A4 expression in the xenograft tumors.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Clinical Proteomics, Plasmid Preparation, Control, Generated, Sequencing, Immunostaining

Journal: Cancers

Article Title: Combination of Wnt/β-Catenin Targets S100A4 and DKK1 Improves Prognosis of Human Colorectal Cancer

doi: 10.3390/cancers14010037

Figure Lengend Snippet: List of differentially expressed Wnt signaling pathway-related factors or target genes upon ectopic expression of S100A4 in HAB92 cells with restored Wnt signaling pathway activity.

Article Snippet: Lyophilized recombinant human DKK1 (rDKK1) protein (R&D Systems, Minneapolis, MN, USA) was dissolved in PBS, supplemented with 1% BSA (Sigma, St. Louis, MO, USA).

Techniques: Expressing, Activity Assay, Activation Assay, Cell Surface Receptor Assay, Migration, Binding Assay, Translocation Assay, Ubiquitin Proteomics

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

Journal: FEBS letters

Article Title: Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

doi: 10.1016/j.febslet.2014.11.023

Figure Lengend Snippet: Fig. 1. Effect of ox-LDL oxidized low-density lipoprotein (ox-LDL) on DKK1 release in macrophages derived from THP-1 cells. (A–C) After stimulation with PMA (160 nM), THP-1 cells differentiated into macrophages were incubated with different concentrations of ox-LDL for various times. Shows western blot (A, B) and RT-PCR (C) analysis of protein and mRNA levels of DKK1 in macrophages. (D) ELISA of DKK1 secretion in cell culture medium. One-way ANOVA with LSD post hoc test, n = 3.

Article Snippet: Recombinant human DKK1 (rDKK1) and human DKK1 ELISA kit were from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: FEBS letters

Article Title: Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

doi: 10.1016/j.febslet.2014.11.023

Figure Lengend Snippet: Fig. 2. The interaction of liver X receptor a (LXRa) and b-catenin is involved in ox- LDL regulation of DKK1. (A) Western blot analysis of siRNA knockdown of b-catenin and cyclinD1 in macrophages differentiated from THP-1 cells. (B) Level of DKK1 regulated by ox-LDL (100 mg/L) with or without b-catenin siRNA. (C, E, G) Cells were pretreated with DMSO or T0901317 (5 lM), GW3965 (1 lM), or 22-(R)- hydroxycholesterol (10 lM), then incubated with 100 mg/L ox-LDL for 6 h. (D, F) Cells were pretreated with GGPP (10 lM), 22-(S)-hydroxycholesterol (10 lM) or ox-LDL (100 mg/L) to mimic the inhibition of LXRa activity by ox-LDL. (H) Western blot analysis of DKK1 with knockdown of LXRa by siRNA. One-way ANOVA with LSD post hoc test, n = 3–4.

Article Snippet: Recombinant human DKK1 (rDKK1) and human DKK1 ELISA kit were from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Knockdown, Incubation, Inhibition, Activity Assay

Journal: FEBS letters

Article Title: Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

doi: 10.1016/j.febslet.2014.11.023

Figure Lengend Snippet: Fig. 3. Effect of DKK1 on foam cell formation in macrophages. (A) Cells were transfected with DKK1 siRNA or scrambled siRNA, then incubated with 100 mg/L ox-LDL. Internalization of ox-LDL was detected by Oil Red O staining. (B) Confocal microscopy of macrophages transfected with DKK1 siRNA, then Dil-ox-LDL (red) added into the cell medium overnight. DAPI was used to stain nuclei. (C) Cells were incubated with recombinant DKK1 (rDKK1; 100 ng/mL) for 12 h, then 100 mg/L ox-LDL was added to examine foam cell formation. (D) Macrophages were pretreated with rDKK1 (100 ng/mL) for 12 h, then incubated with Dil-ox-LDL overnight in the dark. Unpaired t-test, n = 3.

Article Snippet: Recombinant human DKK1 (rDKK1) and human DKK1 ELISA kit were from R&D Systems (Minneapolis, MN, USA).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Recombinant

Journal: FEBS letters

Article Title: Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

doi: 10.1016/j.febslet.2014.11.023

Figure Lengend Snippet: Fig. 4. Wnt/b-catenin mediates the regulation of DKK1 in lipid metabolism of macrophages. (A) Western blot analysis of the effect of DKK1 siRNA on protein expression of DKK1 and total and phosphorylated b-catenin. (B) Western blot analysis of total and phosphorylated b-catenin in cells with rDKK1 treatment and incubated with or without ox-LDL (100 mg/L). (C and D) Oil Red O staining and confocal microscopy of cells transfected with b-catenin siRNA, then ox-LDL or Dil-ox-LDL added into the cell culture medium. One-way ANOVA or unpaired t-test, n = 3.

Article Snippet: Recombinant human DKK1 (rDKK1) and human DKK1 ELISA kit were from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Incubation, Staining, Confocal Microscopy, Transfection, Cell Culture

Journal: FEBS letters

Article Title: Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

doi: 10.1016/j.febslet.2014.11.023

Figure Lengend Snippet: Fig. 5. Wnt and STAT3 pathways mediate the regulation of LOX-1 and ABCA/G1 by DKK1, respectively. Western blot analysis of protein level of LOX-1 in macrophages (A) incubated with rDKK1 (100 ng/mL) for 12 h and (B) with b-catenin siRNA knockdown before adding 100 mg/L ox-LDL. (C and D) Western blot analysis of p- STAT3, total STAT3, ABCA1 and ABCG1 levels in cells pretreated with DMSO or Stattic (10 lM) for 1 h before and during stimulation with rDKK1 and (E) in cells transfected with STAT3 siRNA or negative control, and rDKK1 added into the supernatant. One-way ANOVA with LSD post hoc test, n = 3–6.

Article Snippet: Recombinant human DKK1 (rDKK1) and human DKK1 ELISA kit were from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Incubation, Knockdown, Transfection, Negative Control

Journal: FEBS letters

Article Title: Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

doi: 10.1016/j.febslet.2014.11.023

Figure Lengend Snippet: Fig. 6. An intrinsic mechanism for DKK1 inhibiting lipid accumulation in macro- phages. Ox-LDL increases the levels of DKK1 by decreasing the repression of LXRa on b-catenin. DKK1 activates the STAT3 pathway and blocks the Wnt/b-catenin pathway, which increases the levels of ABCA/G1 and decreases that of LOX-1. This process accelerates lipid efflux and attenuates lipid uptake, for reduced lipid accumulation in macrophages.

Article Snippet: Recombinant human DKK1 (rDKK1) and human DKK1 ELISA kit were from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Developmental cell

Article Title: Lypd6 enhances Wnt/β-catenin signaling by promoting Lrp6 phosphorylation in raft plasma membrane domains.

doi: 10.1016/j.devcel.2013.07.020

Figure Lengend Snippet: Figure 1. Zebrafish lypd6 Is a Wnt/b-Catenin Target Gene that Codes for a GPI-Anchored Plasma Membrane Protein (A) lypd6 whole-mount in situ hybridization (WMISH) showing downregulation in transgenic embryos expressing Axin1 or Dkk1 at gastrula and somitogenesis stages (n [85% epiboly]: Axin1 16/17 embryos, Dkk1 19/19; n [10-somites]: Axin1 21/21, Dkk1 18/18). hs, heat shock. (B) lypd6 expression levels determined by qRT-PCR in hs:Axin1, hs:Dkk1, and hs:Wnt8 transgenic embryos treated as in (A) and shown relative to those in wild- type embryos. Error bars, SEM. (C) Domain structure of N-terminally GFP-tagged wild-type (spGFP-Lypd6) and C-terminally truncated Lypd6 (spGFP-Lypd6DGPI). (D) Localization of spGFP-Lypd6 and spGFP-Lypd6DGPI in enveloping layer (EVL) cells of dome stage zebrafish embryos and in Chinese hamster ovary (CHO) cells. (E) Release of spGFP-Lypd6 and spGFP-GPI from the plasma membrane of HEK293T cells upon treatment with phosphatidylinositol-specific phospholipase C (PiplC). spGFP-Lypd6DGPI and spGFP are detectable in the conditioned media without PiplC treatment. See also Figure S1.

Article Snippet: At 24 hr after transfection with mRFP-GPI, spmCherry-Lypd6, or spmCherry-Lypd6DGPI-CD44 TMD, HEK293T cells were incubated at 4 C for 30 min with binding medium (DMEM, 20 mM HEPES [pH 7.5], 0.1% bovine serum albumin) and subsequently incubated at 4 C for 60minwith bindingmedium supplemented with 200 ng/ml recombinant mouseWnt3a (rWnt3a) or human Dkk1 (rDkk1) (R&D Systems).

Techniques: Clinical Proteomics, Membrane, In Situ Hybridization, Transgenic Assay, Expressing, Quantitative RT-PCR

Journal: Developmental cell

Article Title: Lypd6 enhances Wnt/β-catenin signaling by promoting Lrp6 phosphorylation in raft plasma membrane domains.

doi: 10.1016/j.devcel.2013.07.020

Figure Lengend Snippet: Figure 6. Lypd6 Knockdown or Mislocalization to Nonraft Membrane Domains Shifts Lrp6 Phosphory- lation to These Domains and Inhibits Wnt Signaling (A) pBAR activity in HEK293T cells transfected with Wnt8 (20 ng) plus spGFP-Lypd6 (100 ng) or spGFP-Lypd6DGPI- CD44 TMD (95 ng) or equimolar amounts of spGFP-GPI control. Error bars, SEM. (B) Classes of phenotypes in wnt8-overexpressing embryos injected with wnt8 (20 pg) plus spGFP-lypd6 (150 pg) or spGFP-lypd6DGPI-TfR TMD (140 pg) or equimolar amounts of spGFP-GPI control RNA. spGFP-lypd6DGPI-TfR TMD significantly rescues Wnt8-induced phenotypes. Class 5, hyperdorsalization. (C) spGFP-Lypd6DGPI-CD44 TMD reduces Wnt3a-induced phosphorylation of Lrp6 at S1490 in HEK293T cells assayed at 6 hr poststimulation with Wnt3a CM. (D) lypd6 esiRNA reduces Wnt3a-induced Lrp6 phosphory- lation in HEK293T cells. (E) Fractionation of plasma membrane derived from HEK293T cells treated with Wnt3a into soluble and deter- gent-resistant fractions (DRMs). Endogenous TfR2 (a marker for soluble fractions), Caveolin-1 (Cav1, marking DRMs), P-Lrp6 (S1490), Lrp6, and overexpressed GFP in spGFP- Lypd6 (5 mg) or spGFP-Lypd6DGPI-TfR TMD (4.6 mg) or control spGFP-GPI (2.9 mg) transfected cells were detected by western blotting. (F) Fractionation of plasma membrane derived from HEK293T cells transfected with 2 mg EGFP control or lypd6 esiRNAs and treated with Wnt3a CM into soluble and DRM phases. (G) Imaging and quantification of Wnt3a- or Dkk1-induced Lrp6 speckles in HEK293T cells stably expressing Lrp6- EGFP. Scale bar, 10 mm. See also Figure S5.

Article Snippet: At 24 hr after transfection with mRFP-GPI, spmCherry-Lypd6, or spmCherry-Lypd6DGPI-CD44 TMD, HEK293T cells were incubated at 4 C for 30 min with binding medium (DMEM, 20 mM HEPES [pH 7.5], 0.1% bovine serum albumin) and subsequently incubated at 4 C for 60minwith bindingmedium supplemented with 200 ng/ml recombinant mouseWnt3a (rWnt3a) or human Dkk1 (rDkk1) (R&D Systems).

Techniques: Knockdown, Membrane, Activity Assay, Transfection, Control, Injection, Phospho-proteomics, esiRNA, Fractionation, Clinical Proteomics, Derivative Assay, Marker, Western Blot, Imaging, Stable Transfection, Expressing