Journal: Scientific Reports

Article Title: A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors

doi: 10.1038/s41598-018-36447-4

Figure Lengend Snippet: MSC2504877 is a novel, drug-like, small molecule tankyrase inhibitor. ( A ) Structure of MSC2504877. ( B ) Dose-response curve illustrating the inhibition of recombinant tankyrase (TNKS) or PARP1 with MSC2504877A. Tankyrase activity was assayed using the PARP domain of recombinant human Tankyrase (TNKS) or PARP1 in an ELISA assay. Mean dose response data from three independent experiments is shown. Error bars represent standard error of the mean (SEM). ( C ) Table illustrating IC 50 concentrations obtained for MSC2504877A and two toolbox tankyrase inhibitors. TNKS, TNKS2 and PARP1 activity was determined as in ( B ). ( D ) Western blot illustrating tankyrase stabilisation, suppression of β-catenin and stabilisation of Axin 2 protein levels in COLO320DM (APC mutant) colorectal tumour cells exposed to MSC2504877 for 24 hours as shown. α-Tubulin was used as loading control ( E , F ). Luminex antibody-based detection of AXIN2 and TNKS in COLO320DM cells exposed MSC2504877 in vitro . Dose response data from three independent experiments are shown; error bars represent standard deviations ( G , H ) Luminex detection of AXIN2 and TNKS in COLO320DM xenografts. CB17 SCID mice received 30 mg/kg MSC2504877 via an oral route. At the time points indicated, mice were sacrificed and xenografts recovered. Each data point indicates data from one animal. ( I ) Plasma concentration time profile of MSC2504877 after one single oral dose of 30 mg/kg.

Article Snippet: A monoclonal AXIN2 antibody (R&D Systems #MAB6078) bound to fluorescent carboxybeads was used for isolation of AXIN2 from lysates.

Techniques: Inhibition, Recombinant, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Mutagenesis, Control, Luminex, In Vitro, Clinical Proteomics, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

doi: 10.1074/jbc.m800420200

Figure Lengend Snippet: FIGURE 1. Domain structure of APC and localization of mutant APC proteins in Xenopus A6 cells. A, the domain composition of X. laevis APC (black letters) and its binding partners (red letters). B and C, GFP-fused APC proteins used to analyze subcellular distribution. The results are summarized to the right. D, the localization of full-lengthandofmutatedAPCproteinsinXenopusA6cells.ThecellswerefixedandstainedforGFP(green)and tubulin (MTs, red). Only typical examples are shown. See also supplemental Fig. 1. Bars, 20 m.

Article Snippet: A X. laevis Axin1 cDNA (BC089276, IMAGE:3379167) and a human vinculin cDNA were obtained fromOpen Biosystems and OriGene, respectively.

Techniques: Mutagenesis, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

doi: 10.1074/jbc.m800420200

Figure Lengend Snippet: FIGURE 2. Identification of SAMP motif-binding proteins. A, the Xenopus APC fragment used for yeast two-hybrid screening and the corresponding regions of human and mouse APC. The SAMP (Ser-Ala-Met-Pro) motif is indicated with dots. B, the binding site to the prey proteins was narrowed down by yeast two-hybrid -galactosidase activity detection using APC fragments containing the first SAMP motif (Frag.1) and the last half of APC fragment used for library screening (Frag.2). The results are indicated to the right. C, coprecipitation (IP) of mRFP-fused SAMP binding candidates with GFP-fAPC immobilized to protein A-Sepharose using anti GFP mAb. Axin1 (positive control), DDEF1, DDEF2, and AP-2 mu1 subunit were significantly precipitated with GFP-fAPC. WB, Western blot. Input lanes contain 10% of the cell lysate.

Article Snippet: A X. laevis Axin1 cDNA (BC089276, IMAGE:3379167) and a human vinculin cDNA were obtained fromOpen Biosystems and OriGene, respectively.

Techniques: Binding Assay, Two Hybrid Screening, Activity Assay, Library Screening, Positive Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

doi: 10.1074/jbc.m800420200

Figure Lengend Snippet: FIGURE 3. In vitro binding analysis of the SAMP motif of APC with the SH3 domains. A, the domain structure of Axin1, DDEF1, DDEF2, ARHGAP26, and ARHGAP10. RGS, regulation of G-protein signaling domain; DIX, dishevelled and axin domain; PH, Pleckstrin homology domain; Ank, ankyrin repeats. The SH3 domains of DDEF2 and of ARH- GAP26wereidentifiedasnewSAMPmotif-interactingcandidatesbyyeasttwo-hybridscreening.B,invitrobinding assay. Purified Trx-S-tagged recombinant proteins indicated (0.2 M) were mixed with beads coated with FLAG- SAMP#1viaanti-FLAGantibody(0.2M).BoundproteinswereanalyzedbyWesternblot(WB)analysis.Inputlanes contain 2% of the protein samples used for the binding assay. C, competition between GST-DDEF2-SH3 and Trs-S- Axin1-RGS for in vitro binding to SAMP#1. Immobilized FLAG-SAMP#1 (0.2 M) was incubated with an equal concentration of Trx-S-Axin1-RGS (0.2 M), and the concentration of GST-DDEF-SH3 was increased as indicated. Bound proteins were analyzed by Western blot analysis. Input lanes contain 2% of the protein samples used for binding assay.

Article Snippet: A X. laevis Axin1 cDNA (BC089276, IMAGE:3379167) and a human vinculin cDNA were obtained fromOpen Biosystems and OriGene, respectively.

Techniques: In Vitro, Binding Assay, Purification, Recombinant, Incubation, Concentration Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

doi: 10.1074/jbc.m800420200

Figure Lengend Snippet: FIGURE 6. Association of DDEFs with APC-SAMP in cells. A and B, co-localization of mRFP-fused DDEF1 and DDEF2 with GFP-APC(1641) at the cell edges. Insets are 3 zoom of the boxed areas. C, distribution of endogenous DDEF2 in A6 cells expressing GFP-fAPC. Their detectable co-localization is indicated with arrows. Bars, 20 m (A–C). D, immunoprecipitation (IP) of APC-binding proteins with GFP-APC(1574) and GFP- APC(1641). DDEFs and Axin1 mainly interact with GFP-APC(1641), whereas -catenin and KAP3, positive controlsassociatingwiththeupstreamofSAMPregionofAPC,wereprecipitatedequallywithGFP-APC(1574) and GFP-APC(1641). Note the possible contribution of endogenous APC associating with GFP fusion proteins through their NH2-terminal coiled-coli region. Input lanes contain 100 and 2% of the cell lysates for GFP and for other protein detection, respectively. In B and C, to detect endogenous DDEF1, anti-AMAP1 pAb was used. E, co-sedimentation of DDEF1 and DDEF2 with MTs from A6 cell lysates. The blot of p150glued, a MT-binding protein, is shown as a positive control. Input lanes contain 10% of the cell lysates. WB, Western blot.

Article Snippet: A X. laevis Axin1 cDNA (BC089276, IMAGE:3379167) and a human vinculin cDNA were obtained fromOpen Biosystems and OriGene, respectively.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Sedimentation, Positive Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

doi: 10.1074/jbc.m800420200

Figure Lengend Snippet: FIGURE 7. Expression of APC, Axin1, or DDEF2 affects FA protein paxillin and MTs. A, establishment of A6 stable transfectants. Expressions of GFP- tagged exogenous proteins (GFP-fAPC, 340 kDa (11); GFP-APC(SAMP), 260 kDa; GFP-rAxin, 120 kDa; GFP-DDEF2, 140 kDa) were detected by Western blot (WB) analysis using anti-GFP antibody. Total cell lysates were prepared by lysing the cells cultured for 24 h (5 105 cells/well of 6-well plate) with SDS-sample buffer. For all the proteins, several bands of degrada- tion products are observed. The dots indicate the full-length proteins expressed. B, the phosphorylation status of paxillin at Tyr-31 in the A6 trans- fectants. The cell lysates were subjected to Western blot analysis using anti- paxillin (Tyr-31) antibody. The same blot was stripped and reprobed with anti-paxillin antibody and anti-tubulin antibody (loading control). The levels of paxillin phosphorylation were significantly reduced in GFP-APC(SAMP), GFP-rAxin, and GFP-DDEF2-expressing cells. C, the acetylation status of -tu- bulin in the transfectants. The levels of -tubulin acetylation are reduced in GFP-rAxin and GFP-DDEF2-expressing cells.

Article Snippet: A X. laevis Axin1 cDNA (BC089276, IMAGE:3379167) and a human vinculin cDNA were obtained fromOpen Biosystems and OriGene, respectively.

Techniques: Expressing, Western Blot, Cell Culture, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

doi: 10.1074/jbc.m800420200

Figure Lengend Snippet: FIGURE 8. Distribution of APC proteins, MTs, and FAs in A6 transfectants. A, parental A6 cells and A6 stable transfectants expressing GFP-fAPC or GFP-APC(SAMP) (green), cultured for 24 h (5 105 cells/well of 6-well plate), were fixed and stained for -tubulin (MTs, red) and vinculin (cyan). Insets are the 2.5-magnified images of the boxed areas. Arrows indicate the APC-positive MTs extending to the end of cells. Note that in GFP- APC(SAMP)-expressing cells, MTs were tangled behind the FA-rich areas. In the GFP-fAPC-expressing cells, a large number of FAs was generated throughout the basal cell cortex. Bars, 10 m. B, RFP-DDEF1 or RFP-Axin1 was tran- sientlyoverexpressedinA6transfectantsexpressingGFP-fAPC.RFP-DDEF1wasco-localizedwithGFP-fAPCwithout disturbing the distribution of GFP-fAPC along MTs. RFP-Axin1 formed granular aggregation with GFP-fAPC and removed it from MTs (arrows). In the bottom, yellow lines indicate the outline of a cell expressing RFP-Axin1. In both cases, the formation of FAs was restricted to the cell periphery. Bars, 20 m. C, the numbers of FAs visualized with vinculin staining in parental A6 cells and A6 transfectants (indicated in the figure) were counted and plotted in the graph. Results are the means S.E. For each condition 60 cells were observed.

Article Snippet: A X. laevis Axin1 cDNA (BC089276, IMAGE:3379167) and a human vinculin cDNA were obtained fromOpen Biosystems and OriGene, respectively.

Techniques: Expressing, Cell Culture, Staining, Generated

Journal: Biochemical and biophysical research communications

Article Title: Nuclear AXIN2 represses MYC gene expression

doi: 10.1016/j.bbrc.2013.11.089

Figure Lengend Snippet: Subcellular localization of AXIN2 in established human CRC cell lines and intestinal tissues. (A) Western blot analysis of protein lysates prepared from HEK293 cells that were transfected with pcDNA3-NLS-AXIN2. (B) Western blot analysis of protein lysates prepared from HCT116 and SW480 cells that were transduced with lentiviruses expressing a scrambled sequence (Ctrl.) or four independent shRNAs designed to target AXIN2. (C) Western blot analysis of whole cell [W], cytoplasmic [C], and nuclear [N] lysates prepared from HCT116, SW480, and SW620 cell lines. (D) Immunocytochemical analysis of AXIN2 subcellular localization in HCT116, SW480, and SW620 CRC cell lines. (E) Immunohistochemical analysis of human tissue microarrays prepared from uninvolved colonic mucosa, primary colorectal adenomas, and colorectal tumor metastases. Shown are representative images. Grey and black arrows identify cells with cytoplasmic and nuclear AXIN2 staining, respectively.

Article Snippet: To generate the pcDNA3-NLS-AXIN2 expression vector, NLS-AXIN2 was first amplified using pCMV6-entry-AXIN2 (Origene, RC210931) as the template and the indicated primers ( Table S1 ) in standard PCR reactions containing Phusion polymerase (New England Biolabs).

Techniques: Western Blot, Transfection, Transduction, Expressing, Sequencing, Immunohistochemical staining, Staining

Journal: Biochemical and biophysical research communications

Article Title: Nuclear AXIN2 represses MYC gene expression

doi: 10.1016/j.bbrc.2013.11.089

Figure Lengend Snippet: Nuclear AXIN2 decreases the activity of a Wnt/β-catenin-responsive luciferase reporter. (A) Western blot analysis of whole cell [W], cytoplasmic [C], and nuclear [N] protein lysates prepared from HEK293 cells. (B) Western blot analysis of protein lysates prepared from cytoplasmic [C] and nuclear [N] compartments of HEK293 cells that were transfected with pcDNA3-NLS-AXIN2. (C) Luciferase reporter assays in HEK293 cells transfected with the Wnt-responsive TOPflash reporter or the control FOPflash reporter. Where indicated, cells were co-transfected with plasmids encoding TCF4, β-catenin S45F, and NLS-AXIN2. Data are represented as mean +/− SEM (n = 4, *P < 0.05)

Article Snippet: To generate the pcDNA3-NLS-AXIN2 expression vector, NLS-AXIN2 was first amplified using pCMV6-entry-AXIN2 (Origene, RC210931) as the template and the indicated primers ( Table S1 ) in standard PCR reactions containing Phusion polymerase (New England Biolabs).

Techniques: Activity Assay, Luciferase, Western Blot, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Nuclear AXIN2 represses MYC gene expression

doi: 10.1016/j.bbrc.2013.11.089

Figure Lengend Snippet: β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top, Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom, Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and histone H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.

Article Snippet: To generate the pcDNA3-NLS-AXIN2 expression vector, NLS-AXIN2 was first amplified using pCMV6-entry-AXIN2 (Origene, RC210931) as the template and the indicated primers ( Table S1 ) in standard PCR reactions containing Phusion polymerase (New England Biolabs).

Techniques: Western Blot, Expressing, Immunoprecipitation, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Nuclear AXIN2 represses MYC gene expression

doi: 10.1016/j.bbrc.2013.11.089

Figure Lengend Snippet: AXIN2 alters the chromatin structure at the MYC promoter and decreases MYC expression. (A) Luciferase assays of HCT116 cells transfected with wild-type or mutant MYC 5’ WRE-containing luciferase reporters in the presence or absence of pcDNA3-NLS-AXIN2. (B) Diagram of the MYC gene locus with the MYC 5’ WRE represented as a white box, MYC exons as gray boxes, and introns as thick black lines. The positions of PCR primer sets used to interrogate DNA elements precipitated in the ChIP assays are indicated by opposing arrows. Control (Ctrl.) is a region approximately 287 kb upstream from the MYC transcription start site. (C) ChIP analysis of TCF4 (black bars), β-catenin (gray bars), and AXIN2 (white bars) binding to the MYC 5’ WRE in HCT116 cells. (D) ChIP analysis of AXIN2, β-catenin and H3K4me3, in HCT116 cells transfected with pcDNA3 (Ctrl.) or pcDNA3-NLS-AXIN2. (E) Real-time reverse transcription PCR (qRT-PCR) analysis of MYC expression in control or HCT116 cells expressing NLS-AXIN2. (C-E) Data are represented as mean +/− SEM (n = 4, **P < 0.01, *** P < 0.001).

Article Snippet: To generate the pcDNA3-NLS-AXIN2 expression vector, NLS-AXIN2 was first amplified using pCMV6-entry-AXIN2 (Origene, RC210931) as the template and the indicated primers ( Table S1 ) in standard PCR reactions containing Phusion polymerase (New England Biolabs).

Techniques: Expressing, Luciferase, Transfection, Mutagenesis, Binding Assay, Quantitative RT-PCR