Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: LRP6 expression is frequently up-regulated in a subset of human breast cancer tissues and cell lines. (A) Breast cancer TissueScan Real-Time qPCR array, containing seven normal/Stage 0 cDNAs and 41 human breast cancer cDNAs, was analyzed for LRP6 expression by real-time PCR. Averages of relative LRP6 expression from three independent plates are plotted with clinical status indicated. LRP6 mRNA levels are markedly up-regulated in a subset of human breast cancer tissues. #Samples with elevated HER2 transcripts. (B and C) Breast cancer tissue microarray was used for IHC staining of LRP6. (B) Representatives of LRP6 staining in normal and malignant breast tissue are shown. LRP6 antibody (C-term T1546, Abgent), which specifically recognizes human LRP6, was used for IHC staining. (C) The quantification of LRP6 IHC staining was determined from three independent experiments. Staining intensity was scored as absent (0), weak (1), moderate (2), or strong (3). Four observations were made on each slide by independent investigators, and a mean score was recorded. (D) Expression of LRP6 in human mammary epithelial cell (MCF-10A) and indicated breast cancer cell lines analyzed by Western blot analysis. *P < 0.05; **P < 0.01.

Article Snippet: * P < 0.05; ** P < 0.01. ( F ) Gross examination of xenograft tumors. ( G ) Levels of LRP6 and Wnt target gene expressions ( Cyclin D1, c-Myc , and Axin2 ) in control and LRP6-KD xenograft tumors detected by Western blot analysis. ( H ) Immunohistochemical analysis of LRP6 level in control and LRP6-KD xenograft tumors with anti-LRP6 antibody (Abgent). (Scale bars, 50 μm.) ( I ) Western blot analysis showing total and free β-catenin in LRP6-KD tumors were decreased.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Microarray, Immunohistochemistry, Staining, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: Knockdown of LRP6 in MDA-MB-231 breast cancer cells decreases Wnt signaling, breast cancer cell viability, proliferation, and colony formation. MDA-MB-231 cells were transduced with lentivirus expressing control or LRP6 shRNA. Cells were then subjected to the indicated analysis 48 h postinfection. (A) Western blot and densitometric analysis show that both LRP6 shRNAs reduce LRP6 expression in MDA-MB-231 cells compared with control shRNA. (B) LRP6 down-regulation suppressed Wnt signaling examined by free β-catenin pull-down (Left) and TOPFlash reporter assays in the absence (Center) and presence (Right) of Wnt3a ligands. (C) Quantitative real-time PCR shows that expression of Wnt target genes (Cyclin D1, c-Myc, and Axin2) is down-regulated in cancer cells expressing LRP6 shRNA2. GAPDH was included as a control gene. (D) Knockdown of LRP6 decreased cell viability assessed by MTT assay. (E) Proliferation of breast cancer cells expressing LRP6 shRNA2 was suppressed by ∼50% as measured by BrdU incorporation. (F) Soft agar colony formation assay demonstrating reduced colony formation when LRP6 expression was knocked down. Data are mean ± SD from three independent experiments. *P < 0.05; **P < 0.01.

Article Snippet: * P < 0.05; ** P < 0.01. ( F ) Gross examination of xenograft tumors. ( G ) Levels of LRP6 and Wnt target gene expressions ( Cyclin D1, c-Myc , and Axin2 ) in control and LRP6-KD xenograft tumors detected by Western blot analysis. ( H ) Immunohistochemical analysis of LRP6 level in control and LRP6-KD xenograft tumors with anti-LRP6 antibody (Abgent). (Scale bars, 50 μm.) ( I ) Western blot analysis showing total and free β-catenin in LRP6-KD tumors were decreased.

Techniques: Transduction, Expressing, shRNA, Western Blot, Real-time Polymerase Chain Reaction, MTT Assay, BrdU Incorporation Assay, Soft Agar Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: shRNA-resistant LRP6 and CA β-catenin rescue Wnt signaling and cell growth in MDA-MB-231 cells. (A and B) MDA-MB-231 cells expressing control or LRP6 shRNA were transfected with vector control or shRNA-resistant LRP6 (LRP6-Res). (A) Levels of LRP6 expression were examined by Western blot analysis. (B) Cells were then treated with L or Wnt3a CM. Expression of LRP6-Res in MDA-MB-231 cells restored Wnt signaling, detected by TOPFlash reporter assay. (C) Expression of LRP6-Res in MDA-MB-231 cells rescued cell growth detected by MTT assay. (D–F) MDA-MB-231 cells expressing control or LRP6 shRNA were transduced with retrovirus expressing IRES-GFP vector control or CA β-catenin. (D) Free β-catenin levels were analyzed by GST–E-cadherin pull-down. (E) CA β-catenin promoted Wnt activation independent of Wnt3a ligand. (F) CA β-catenin expression restored breast cancer cell growth determined by MTT assay. All results are the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01.

Article Snippet: * P < 0.05; ** P < 0.01. ( F ) Gross examination of xenograft tumors. ( G ) Levels of LRP6 and Wnt target gene expressions ( Cyclin D1, c-Myc , and Axin2 ) in control and LRP6-KD xenograft tumors detected by Western blot analysis. ( H ) Immunohistochemical analysis of LRP6 level in control and LRP6-KD xenograft tumors with anti-LRP6 antibody (Abgent). (Scale bars, 50 μm.) ( I ) Western blot analysis showing total and free β-catenin in LRP6-KD tumors were decreased.

Techniques: shRNA, Expressing, Transfection, Plasmid Preparation, Western Blot, Reporter Assay, MTT Assay, Transduction, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy

doi: 10.1073/pnas.0911220107

Figure Lengend Snippet: Down-regulation of LRP6 significantly inhibits breast tumor growth in vivo. MDA-MB-231 cells (pooled clones) stably expressing control or LRP6 shRNA were injected s.c. into female BNX immunocompromised mice. Tumors initiated from 5 × 105 or 2 × 106 cancer cells were indicated as Low # or High #, respectively. Low #, thoracic pair; High #, caudal pair; control tumors, left; LRP6-KD tumors, right. Tumor growth was monitored over time for 3 weeks by in vivo bioluminescent imaging and caliper measurements. (A) Representative bioluminescence images over time from the same mouse bearing MDA-MB-231 xenografts. (B and D) Growth of tumors over time for control and LRP6-KD xenografts is shown as fold changes of bioluminescence photon flux values over initial value (1 day postinjection). Data are mean ± SEM (six animals, four tumors each) from two independent experiments. (C and E) Tumor volume was monitored for 3 weeks by caliper measurement. (LxWxD). *P < 0.05; **P < 0.01. (F) Gross examination of xenograft tumors. (G) Levels of LRP6 and Wnt target gene expressions (Cyclin D1, c-Myc, and Axin2) in control and LRP6-KD xenograft tumors detected by Western blot analysis. (H) Immunohistochemical analysis of LRP6 level in control and LRP6-KD xenograft tumors with anti-LRP6 antibody (Abgent). (Scale bars, 50 μm.) (I) Western blot analysis showing total and free β-catenin in LRP6-KD tumors were decreased.

Article Snippet: * P < 0.05; ** P < 0.01. ( F ) Gross examination of xenograft tumors. ( G ) Levels of LRP6 and Wnt target gene expressions ( Cyclin D1, c-Myc , and Axin2 ) in control and LRP6-KD xenograft tumors detected by Western blot analysis. ( H ) Immunohistochemical analysis of LRP6 level in control and LRP6-KD xenograft tumors with anti-LRP6 antibody (Abgent). (Scale bars, 50 μm.) ( I ) Western blot analysis showing total and free β-catenin in LRP6-KD tumors were decreased.

Techniques: In Vivo, Clone Assay, Stable Transfection, Expressing, shRNA, Injection, Imaging, Western Blot, Immunohistochemical staining

Journal: Oncotarget

Article Title: Rottlerin as a novel chemotherapy agent for adrenocortical carcinoma

doi: 10.18632/oncotarget.15221

Figure Lengend Snippet: ( A ) The wound healing assay showed that rottlerin reduced the cell migration rate of SW-13 cells compared with the control and DMSO groups. ( B ) WB assays of β-catenin, LRP6, and p-LRP were performed in NCI-H295R and SW-13 cells. The results showed that rottlerin down-regulated the expression of β-catenin, LRP6, and p-LRP in a dose-dependent manner. ( C ) The 24 h migration of SW-13 decreased after rottlerin treatment compared with the control and DMSO-treated groups (* p < 0.05).

Article Snippet: The antibodies used for Western blotting and immunohistochemistry staining were as follows: anti-LRP6 and anti-p-LRP6 antibodies were purchased from Affinity Bioscience (Cincinnati, OH, USA); and anti-β-catenin antibody was purchased from ABCAM (San Francisco, CA, USA).

Techniques: Wound Healing Assay, Migration, Control, Expressing

Journal: Oncotarget

Article Title: Rottlerin as a novel chemotherapy agent for adrenocortical carcinoma

doi: 10.18632/oncotarget.15221

Figure Lengend Snippet: The positive expressing proteins appeared brown in color. The results showed that rottlerin down-regulated the expression of β-catenin, LRP6, and p-LRP compared with the control and DMSO-treated groups (* p < 0.0 5 ) in vivo . The suppression was stronger in the higher dose-treated group (rottlerin 4 mg/kg) than the lower dose-treated group (rottlerin 2 mg/kg; ** p < 0.05).

Article Snippet: The antibodies used for Western blotting and immunohistochemistry staining were as follows: anti-LRP6 and anti-p-LRP6 antibodies were purchased from Affinity Bioscience (Cincinnati, OH, USA); and anti-β-catenin antibody was purchased from ABCAM (San Francisco, CA, USA).

Techniques: Expressing, Control, In Vivo

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis

doi: 10.1038/gim.2016.10

Figure Lengend Snippet: Overview of LRP6 variants identified with MIP screen in OFC and TA patients vs. controls All variants were coding or canonical splice site variants with a population frequency of <0.1% (based on dbSNP142 and an in-house database with >5,000 samples), ≤3 samples in this study with the same variant and a ‘GATK quality by depth’ of >1,000, the latter was based on previous MIP data and extensive validations by Sanger sequencing showing low false positive rates and high sensitivity. Minimal average coverage over all MIPs of included samples was 100-fold. Most unique and rare non-synonymous variants reported here in cases have been validated by Sanger sequencing.

Article Snippet: To check the expression of Lrp6 in mouse developing tooth and palate, we generated paraffin sections of E13 and E15 mouse embryo heads and stained them with anti-Human LRP6 polyclonal antibody (cat. nr.orb18907, Progen, Sanbio, the Netherlands) counterstained with Hematoxylin.

Techniques: Variant Assay, Sequencing

Journal: Journal of Cell Science

Article Title: Stability elements in the LRP6 cytoplasmic tail confer efficient signalling upon DIX-dependent polymerization

doi: 10.1242/jcs.067546

Figure Lengend Snippet: Phosphorylation of DIX>ctail depends on a polymerization-competent DIX domain. (A) Western blots of total lysates of HEK293 cells transfected with wt and mutant DIX>ctail, showing phospho-S1490 levels (top) relative to total expression levels of wt and mutant DIX>ctail (bottom panels; re-probing of the blots shown above). Note the reductions of phospho-S1490 (indicated by arrows) in the polymerization-deficient mutants (M4>ctail and M4>ctailΔA behave the same as their M2-mutant counterparts; not shown), and the lack of a signal in m1 (bearing an S1490A substitution). (B-E) HeLa cells expressing DIX>ctail wt and deletion mutants, immunostained with the phospho-specific Tp1479 antibody, revealing strong punctate staining for DIX>ctail and DIX>ctailΔB, but complete absence of staining for M2>ctail and M2>ctailΔB. Scale bars: 10 μm.

Article Snippet: Immunofluorescence and western blot analysis Cells were fixed and stained with the following antibodies, as described previously ( Schwarz-Romond et al., 2007b ): anti-LRP6 (Santa Cruz); anti-Dvl2 (H-75, Santa Cruz; ); anti-pLRP6 Ser1490 (Cell Signalling Technologies) ( Zeng et al., 2005 ); anti-LRP6 Tp1479 ( Davidson et al., 2005 ) (a kind gift from Christof Niehrs); anti-β-catenin (C2206, Sigma); anti-GFP (G1544, Sigma); mouse anti-FLAG (F3165, Sigma); rabbit anti-FLAG (F7425, Sigma; ); anti-Wingless, anti-Sxl (Developmental Studies Hybridoma Bank); antibodies against vesicle markers (supplementary material Fig. S2) were previously described ( Schwarz-Romond et al., 2005 ).

Techniques: Phospho-proteomics, Western Blot, Transfection, Mutagenesis, Expressing, Staining

Journal: Journal of Cell Science

Article Title: Stability elements in the LRP6 cytoplasmic tail confer efficient signalling upon DIX-dependent polymerization

doi: 10.1242/jcs.067546

Figure Lengend Snippet: Stability elements in the LRP6 ctail are essential for its efficient signalling activity. (A-E) Live-cell imaging of HeLa cells expressing (A) Dvl2-GFP or (C) DIX>ctail, subjected to FRAP analysis; relative fluorescence intensities were measured after bleaching of >10 single puncta (boxed in red) by short laser pulses (at T=4 seconds), and plotted against time (B,D,E). Fluorescence recovery is observed for every single Dvl2-GFP punctum (A,B), but not for any of the puncta in wt DIX>ctail or single deletion mutants (B,C). (D,E) FRAP analysis of multiple DIX>ctailΔAB puncta (D), exhibiting variable recovery (see also text), and of DIX>ctailΔABplus puncta (E), exhibiting complete stability, restored by the KGTYFP epitope. (F) TOPFLASH assays in HEK293 cells, revealing restoration of signalling activity of the double-deletion by the KGTYFP epitope (values are directly comparable to those in Fig. 2F and Fig. 4B). Inset: protein expression levels. (G) HeLa cells expressing DIX>ctailΔABplus, immunostained with anti-Tp1479, showing that T1479 within the KGTYFP epitope remains unphosphorylated in the DIX>ctailΔABplus puncta (see also Fig. 5B-E), probably because of the lack of a −3 priming residue.

Article Snippet: Immunofluorescence and western blot analysis Cells were fixed and stained with the following antibodies, as described previously ( Schwarz-Romond et al., 2007b ): anti-LRP6 (Santa Cruz); anti-Dvl2 (H-75, Santa Cruz; ); anti-pLRP6 Ser1490 (Cell Signalling Technologies) ( Zeng et al., 2005 ); anti-LRP6 Tp1479 ( Davidson et al., 2005 ) (a kind gift from Christof Niehrs); anti-β-catenin (C2206, Sigma); anti-GFP (G1544, Sigma); mouse anti-FLAG (F3165, Sigma); rabbit anti-FLAG (F7425, Sigma; ); anti-Wingless, anti-Sxl (Developmental Studies Hybridoma Bank); antibodies against vesicle markers (supplementary material Fig. S2) were previously described ( Schwarz-Romond et al., 2005 ).

Techniques: Activity Assay, Live Cell Imaging, Expressing, Fluorescence, Residue