Endogenous expression of LRP5Δ in MCF7 breast cancer cells is required for accumulation of non-phosphorylated active β-catenin and transcriptional activation by β-catenin.

<p>(A) PCR of MCF7 cDNA as in panel A. sHPT-1 parathyroid cDNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004243#pone.0004243-Bjrklund1" target="_blank">[26]</a> was used as marker. Direct sequencing confirmed the in-frame deletion of 142 amino acids (Δ666–809). (B) Specificity and efficiency of siRNAs transiently transfected to MCF7 cells. siLRP5wt is directed to wild type transcripts, siLRP5Δ to the truncated transcript, and siLRP5tot to both transcripts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004243#pone.0004243-Bjrklund1" target="_blank">[26]</a>. Quantitative real-time PCR of both LRP5 transcripts (LRP5tot, left panel) and immunoprecipitation and Western blot analysis of LRP5 (right panel). (C) Western blot analysis of non-phosphorylated active β-catenin after siRNA transfection. (D) Transient cotransfections of TOPFLASH/FOPFLASH or pTOPGlow/pFOPGlow TCF/β-catenin reporter, the CMV-LacZ reference plasmid (left panel), and the indicated siRNAs (right panels) to MCF7 cells. FOPFLASH and pFOPGlow contain mutated binding sites for TCFs, while TOPFLASH and pTOPGlow do not. Luciferase activities were normalized to β-galactosidase activities. The siRNAs displayed no effect on pFOPGlow (not shown) and FOPFLASH reporter activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004243#pone.0004243-Bjrklund1" target="_blank">[26]</a>.</p

Endogenous expression of LRP5Δ in MCF7 breast cancer cells is required for transcriptional activation of DKK1 by β-catenin.

<p>(A) Transient cotransfections of DKK1 promoter luciferase constructs WT short or TBE3,4mut short <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004243#pone.0004243-Niida1" target="_blank">[38]</a> and CMV-LacZ to MCF7 cells (left panel). WT short, CMV-LacZ, and the indicated siRNAs were transfected (right panel). (B) Endogenous DKK1 mRNA expression in siRNA transfected MCF7 cells as indicated. (C) Transient cotransfections of TOPFLASH/FOPFLASH (left panel) or pTOPGlow/pFOPGlow (right panel) and CMV-LacZ to the indicated number of plated MCF7 cells.</p

Expression of LRP5Δ in breast tumor specimens of different cancer stages.

<p>Expression of LRP5Δ in breast tumor specimens of different cancer stages.</p

The LRP5Δ receptor is expressed in breast tumors.

<p>Accumulation of active β-catenin. (A) PCR analysis of cDNA from normal breast tissue (N1–N4) and primary breast cancer (T1–T19). Primers were located in exons 9 and 13 of LRP5 as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004243#pone.0004243-Bjrklund1" target="_blank">[26]</a>. The PCR reaction is not quantitative as the primers compete for the two fragments. A total of sixty-two LRP5 truncated fragments (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004243#pone-0004243-t001" target="_blank">Table 1</a>) were directly sequenced and all contained the same in-frame deletion of 142 amino acids (Δ666–809). (B) Immunoprecipitation and Western blot analysis of LRP5. Tissue numbering corresponds to panel A. Transiently expressed LRP5wt and LRP5Δ shown as markers. (C) Western blot analysis of non-phosphorylated active β-catenin. Tissue numbering as in panel A.</p

Tumor incidence in <i>Ntv-a Arf</i>-/- mice injected with PDGF-B+X (P+X) or PDGF-B+HRG (P+H) RCAS viruses.

<p>Tumor incidence in <i>Ntv-a Arf</i>-/- mice injected with PDGF-B+X (P+X) or PDGF-B+HRG (P+H) RCAS viruses.</p

Presence and expression of viral transduced PDGF-B and HRG in tumors.

<p>(<b>A</b>) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). (<b>B</b>) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).</p

HRG had no effect on primary glial cell proliferation.

<p>(<b>A</b>) Primary glial cells were infected with RCAS-eGFP, RCAS-HRG or RCAS-PDGFB-HA and expression of the viral transduced proteins was analyzed with immunocytochemistry. The infection efficiency was similar in all conditions. (<b>B</b>) Proliferation assay on infected cells showed no effect of HRG compared to control cells on glial cell proliferation at day 7. Curves show the mean (±SEM) from three independent experiments for HRG and eGFP, and two independent experiments for PDGF-B.</p

Distribution of tumor malignancy grades.

<p>(<b>A</b>) Distribution of tumor grades (II–IV) in PDGF-B+X (P+X) and PDGF-B+HRG (P+H) injected <i>Ntv-a Arf-/-</i> mice. * p<0,05 (<b>B</b>) Distribution of low grade (II) versus malignant (III+IV) glioma. *p<0,05.</p

RCAS-HRG infected DF-1 cells could produce functional HRG.

<p>(<b>A</b>) Viral produced HRG protein could be detected by western blot in conditioned media from DF-1 cells infected with RCAS-HRG but not from RCAS-PDGFB or RCAS-eGFP infected cells. Purified HRG was used as a positive control (+). (<b>B</b>) Viral produced HRG could significantly inhibit migration of HUVEC cells towards VEGF. t-test, ** p<0.01, *** p<0.001.</p

Tumor volume is increased in RT2/HRG^−/− compared to RT2/HRG^+/+ mice.

<p>Tumors were dissected and measured. Tumor volumes were calculated by the formula ((π/6)×width²×length). Each dot represents the summarized tumor volume in mm³ from one mouse at (A) 12 weeks of age (n^+/+ = 16; n^−/− = 16) and at (B) 15 weeks of age (n^+/+ = 11; n^−/− = 12). Statistical analyses were performed with a two-tailed Mann-Whitney test, vertical bars represent standard deviation, * p≤0.05.</p