Behavior of the different proteins of the cadherin–catenin complex upon SIP1 induction

<p><b>Copyright information:</b></p><p>Taken from "SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions"</p><p>Nucleic Acids Research 2005;33(20):6566-6578.</p><p>Published online 24 Nov 2005</p><p>PMCID:PMC1298926.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Immunofluorescence microscopy of non-induced and induced DLD1Tr21/WTSIP1 cells using antibodies specific for adherens junction components. E-cadherin as well as αE-catenin, p120ctn and β-catenin became nearly undetectable at cell–cell contacts in the SIP1-induced cells. () Western blot analysis of the non-induced and induced DLD1Tr21/WTSIP1 cell line. E-cadherin and αE-catenin were downregulated at the protein level in the SIP1-expressing cells. β-catenin protein levels were unaltered in the SIP1-induced compared to non-induced cells. Protein expression of p120ctn isoform 1 was upregulated and that of isoform 3 downregulated after SIP1 induction (: addition of Dox every 2 days, : washing away Dox from the cell culture medium)

Expression analysis of the <i>NBPF</i> gene family in neuroblastoma cell lines and tumors.

<p>(A) Real-time quantitative RT-PCR analysis of the <i>NBPF</i> gene family in a panel of neuroblastoma cell lines. Four different amplicons on the <i>NBPF</i> cDNA were chosen, of which A and C can have several copies in a single <i>NBPF</i> transcript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002207#pone.0002207-Vandepoele2" target="_blank">[27]</a>. The top panel shows the location of these amplicons in the standard <i>NBPF1</i> transcript. Amplicon A is present in two copies in this particular transcript. Expression levels were normalized to four reference genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002207#pone.0002207-Vandesompele1" target="_blank">[19]</a>, and the lowest value was set to 1. (B) Neuroblastoma cell lines were grouped according to the <i>NBPF1</i> status (heterozygous <i>NBPF1</i> deletion or not). For all amplicons, this resulted in a median expression level that was lower in cell lines with <i>NBPF1</i> loss, and this difference was significant for amplicons A, B and D (<i>p</i><0.05), but not for amplicon C (<i>p</i> = 0.13). (C) Meta-analysis of the <i>NBPF</i> expression level in neuroblastoma tumors using the Oncomine database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002207#pone.0002207-Rhodes1" target="_blank">[32]</a>. The reporter used in the array experiment was derived from <i>NBPF1</i>, but due to the high sequence identity among the <i>NBPF</i> paralogs, this analysis also represents the expression level of all <i>NBPF</i> genes. Significantly lower levels of <i>NBPF</i> transcripts are observed in tumors that relapse within five years.</p

Induced expression of <i>NBPF1</i> in human tumor cells impairs colony formation in soft agar.

<p>(A) DLD1Tr21/NBPF1 cells express flag-tagged NBPF1 in the cytoplasm, but only when Dox is added to the medium (left panels). DAPI staining was used to visualize nuclei (right panels). (B) DLD1Tr21/NBPF1 and DLD1Tr21/Mock cells were seeded in soft agar in six-well plates in the absence or presence of Dox and then incubated for 2 weeks. NBPF1 induction induces a severe decrease in the ability to form colonies on soft agar. Pictures in the top panel were taken at a 0.8×magnification and those in the bottom panel at a 4×magnification. Scale bars indicate 200 μm. (C) Quantification of the soft agar colonies (mean±standard deviation for two experiments). Only colonies larger than 100 μm were counted. Induction of NBPF1 expression by adding Dox results in a severe decrease in the number of colonies in comparison to the control setting (Mock) or the non-induced (-Dox) cells.</p

Expression analysis of <i>NBPF1</i> in a panel of 32 neuroblastoma cell lines.

<p>Expression levels were normalized to two reference genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002207#pone.0002207-Vandesompele1" target="_blank">[19]</a>, and the lowest value was set to 1. Inset: relative expression levels of <i>NBPF1</i> in neuroblastoma cell lines with normal <i>NBPF1</i> status (grey; NO) or with a heterozygous deletion of the <i>NBPF1</i> locus (white; YES). Significantly lower <i>NBPF1</i> expression is observed in cell lines with <i>NBPF1</i> loss (p<0.05).</p

Genomic overview of the translocation breakpoints of the der(1) and der(17) chromosomes.

<p>Schematic representation of the normal chromosome 1p36 and 17q12 regions is shown in the central box. On chromosome 1 (black bars) both translocation breakpoints are located in intronic regions of the <i>NBPF1</i> gene, which is only partially shown (indicated by the dotted line). The breakpoint of the der(1) chromosome is located in a LINE repeat, shown as a black box labeled L1-PA4 on the genomic region. The black boxes below the genomic sequence represent the exons of the <i>NBPF1</i> gene, with the exon type <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002207#pone.0002207-Vandepoele2" target="_blank">[27]</a> indicated under each box. The translocation resulted in deletion of 5,215 bp. The translocation breakpoint on chromosome 17 (white bars) is located in an <i>ACCN1</i> intron and disrupts isoform 2 but leaves isoform 1 intact. Transcription starts are indicated by angled arrows. The breakpoint is located in a LINE repeat, shown as a white box labelled L2 on the genomic region. White boxes below the genomic sequence represent the exons of the <i>ACCN1</i> gene. On the der(1) chromosome (top), the <i>NBPF1</i> gene is disrupted, giving rise to two chimeric cDNAs (shown as NBPF1-CHI, with the grey box representing the chromosome 17-derived fusion partners). The promoter region and first exon of <i>ACCN1</i> isoform 2 are translocated to the der(1) chromosome, but no chimeric transcripts have been isolated yet (shown with the question mark and dotted line). On the der(17) chromosome (bottom), <i>ACCN1</i> isoform 1 is unaffected by the translocation, but as the promoter and first exon of isoform 2 are translocated, isoform 2 is probably no longer expressed (shown by the dotted line and question mark). The last three <i>NBPF1</i> exons (shown as exons of types 11, 12, and 14) are translocated to the der(17) chromosome. Arrows on the genomic sequence represent promoter sequences, and arrowheads in the transcripts show the orientation of the genes; tel, telomeric end; cen, centromeric end. The figure is not drawn to scale.</p

Cloning of the translocation breakpoints of the constitutional t(1;17) translocation.

<p>(A) Schematic overview of the sequence of a cosmid contig spanning the breakpoint <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002207#pone.0002207-VanRoy3" target="_blank">[18]</a>. Nine repeat-free probes (white boxes) were used in Southern hybridizations. Recognition sites for <i>Pvu</i>II (P) and <i>Eco</i>RI (E) are shown. (B) Southern blot analysis of genomic DNA of human-hamster cell hybrids containing either the der(1) (hybrid 32-7A) or the der(17) chromosome (hybrid 32-2F53VIII). Left panel: hybridization of probe 6 to <i>Pvu</i>II-digested genomic DNA yielded a band of ∼4 kbp in the 32-2F53VIII cell line (arrow) but not in the control samples. Right panel: hybridization of probe 9 to <i>Eco</i>RI-digested genomic DNA showed an additional band of ∼17 kbp in the 32-7A cell line (arrow) but not in the control sample. A/3: parental hamster cell line; normal: normal human genomic placenta DNA. (C) Schematic overview of a fragment of derivative chromosome 17. Two primers located in probe 6 (GSP1 and GSP2) were used in combination with adaptor primers to amplify breakpoint-spanning fragments from genomic DNA digested with <i>Bam</i>HI (B), <i>Dra</i>I (D), <i>Eco</i>RV (EV), <i>Pvu</i>II (P) or <i>Stu</i>I (S). All the resulting fragments contained sequences derived from chromosome 1, with an overlap of 3,134 bp. Seven nucleotides of unknown origin are inserted at the breakpoint site. The location of the t(1;17) translocation breakpoint is indicated by a vertical double-pointed arrow. White boxes represent chromosome 17 sequences, and black boxes represent chromosome 1 sequences. (D) Schematic overview of a fragment of derivative chromosome 1. Two primers located in or nearby probe 9 (GSP3 and GSP4, respectively) were used in combination with adaptor primers to amplify breakpoint-spanning fragments from genomic DNA digested with <i>Dra</i>I (D), <i>Eco</i>RV (EV), <i>Pvu</i>II (P) or <i>Stu</i>I (S). All the resultant fragments contained sequences derived from chromosome 1, with an overlap of 3,845 bp. Four nucleotides of unknown origin are inserted at the breakpoint site. The translocation breakpoint and sequences are indicated as in panel (C).</p

Oncomine expression analysis of <i>ACCN1</i> in neuroblastoma tumors.

<p>Significantly weaker <i>ACCN1</i> expression is observed in tumors with <i>MYCN</i> amplification (MNA), in higher stage tumors (stage 3+4), and in tumors with 1p36 loss of heterozygosity (1p36 LOH), but not in tumors with 11q23 LOH.</p

Cloning of the constitutional t(1;17) translocation breakpoints in the cell hybrids 32-2F53VIII and 32-7A.

<p>Nucleotide sequences of the der(17) (A) and der(1) (B) regions flanking the translocation breakpoints. (C) Compilation of normal and derivative sequences at the breakpoint sites (NCBI Build 36.1) showing complete preservation of chromosome 17 sequences but not of chromosome 1 sequences.</p

Primary antibodies.

<p>Primary antibodies.</p

E-cadherin stabilization by p120ctn is required for proper polarization and cell–cell adhesion by EB cells.

<p><b>(A)</b> Confocal pictures of p120ctn and E-cadherin stainings of control and p120ctn^-/-;AL^tg/+ mESCs, and of p120ctn^-/-;AL^tg/+ mESCs expressing various rescue constructs from the R26 promoter, as indicated by the names above each of the images. An 2.9-fold magnified image is shown below each picture. Scale bars: 25 μm. <b>(B)</b> TEM of DIV12 EBs, control or p120ctn^-/-;AL^tg/+ EBs, or p120ctn^-/-;AL^tg/+ EBs expressing various rescue constructs from the R26 promoter, as indicated. Blue boxes depict areas with mature junctions while blue arrows denote areas of minimal cell–cell adhesion. Black scale bars: 10 μm; white scale bars: 1 μm.</p