Expression of Snail1 in tumor and stroma according to tumor stage.

<p>Snail1 immunoreactivity was determined in the stromal or carcinoma cells corresponding to colorectal tumours classified in the different stages. According to Snail1 expression, tumours were classified as presenting Snail1 expression both in the tumour and stroma (T+/S+), just in the tumour (T+/S−), just in the stroma (T−/S+) or not present in either of these compartments (T−/S−).</p

Specific survival of stage I, II and III colon tumour patients according to Snail1 expression in the stroma.

<p>The presence of Snail1 in the stroma of stage I, II and III tumours is represented as continuous lines; dotted lines correspond to stroma-negative tumours. In the lower left panel, expression of Snail1 in the stroma was considered as low or high according to the criteria indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005595#s4" target="_blank">Methods</a>. The significance is indicated in each category.</p

Kaplan-Meier specific survival curves for colon carcinoma patients according to Snail1 expression in the stroma.

<p>Discontinuous line represents negative Snail1 immunoreactivity; continuous line, Snail1 positive immunoreactivity. The graphics compare tumours where expression of Snail1 was observed in the stroma with respect to negative ones, regardless of the immunoreactivity in the tumour (left); and with only reactivity in the stroma with respect to negative biopsies (right). The p values are indicated.</p

Characteristics of 162 patients with colorectal cancer.

<p>Characteristics of 162 patients with colorectal cancer.</p

Nuclear Snail1 protein expression in colon carcinomas.

<p>Expression of Snail protein was determined as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005595#s4" target="_blank">Methods</a> in samples corresponding to colon carcinomas using MAb EC3. Micrographs of several representative stained sections are shown. Panels A–E corresponded to tumours considered positive only in the stroma; panel F, just in the tumour, and panels G–P; in both compartments. The arrow in panel H labels a cell that cannot be clearly classified as tumoral or stromal. In panel O the arrow points at a cell entering a vessel. Bars indicate magnification.</p

pPS1, and not npPS1, is responsible for down-regulation of E-cadherin levels.

<p>(A) MEF PS (+/+) cells were incubated with 5 µM γ-secretase inhibitor L-685,458 for 24 h. 50 µg of MEFs PS (−/−) and PS (+/+) total cell extracts were analyzed by SDS-PAGE and Western blot with the indicated antibodies. (B) MEF PS (−/−) and PS (+/+) cells were incubated with 25 µg/ml cycloheximide for the indicated time periods. 10 µg of MEF PS (−/−) and 60 µg of MEF PS (+/+) total cell extracts were analyzed by Western blot with anti-E-cadherin, anti-PS1 (N-terminus) and anti-β-actin-specific antibodies. Autoradiograms were scanned; the values obtained for E-cadherin were referred to the initial time. (C) MEF PS (−/−) cells were cotransfected with 2.5 µg of pECFPN1-E-cadherin and 2.5 µg of pcDNA3.1-<i>Myc/His</i>-PS1 (npPS1) or pcDNA3.1-PS1 (pPS1) plasmids. 24 h after transfection, 5 µM γ-secretase inhibitor L-685,458 was added to the medium for 24 h. Percentage of transfection was estimated to be approximately 80%, determining the number of CFP-positive cells. 50 µg of total cell extracts were analyzed by Western blotting with the indicated antibodies. At the right, the ectopic E-cadherin levels of three different experiments were quantified and represented with respect to control (no expression of either pPS1 or npPS1). Values are the average +/− S.D. of these experiments.</p

pPS1 and npPS distinctly affect the expression of β-catenin·Tcf-4-dependent transcriptional targets.

<p>MEF PS (−/−) cells were cotransfected with 10.5 µg of either pcDNA3.1-PS1 (pPS1), pcDNA3.1-<i>Myc/His-</i>PS1 (npPS1) or empty vector and 1.5 µg pBABE-puro hrGFP. After 48 h, transfected cells were selected with 2.5 µg/ml Puromycin for 48 h and protein extracts or RNAs were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004080#s2" target="_blank"><i>Material and Methods.</i></a> (A) 25 µg of total cell extracts were analyzed by SDS-PAGE and Western blot with antibodies against PS1 (1–65), c-myc and β-actin as a control. 10⁵ transfected cells were seeded and cell proliferation analyzed by counting the number of cells every 24 h. The reduction in the proliferation rate of pPS1 and npPS1 transfected cells is shown referred to the proliferation rate of cells transfected with the empty vector. (B) RNAs were obtained from the three transfectants populations as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004080#s2" target="_blank">Methods</a> and the levels of the indicated transcripts analyzed by quantitative RT-PCR. The panel shows the relative expression of these genes with respect to the control (average +/− S.D. of three determinations carried out in triplicate).</p

The CTF2 fragment of E-cadherin interacts with CBP.

<p>(A) GST-E-cadh-CTF2 and GST-N-cadh-CTF2 were phosphorylated with CKIδ, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004080#s2" target="_blank"><i>Material and Methods</i></a>. 20 pmols of non-phosphorylated or phosphorylated GST-cadh-CTF2 proteins, or GST as a control, were incubated with 1.2 mg of MEF PS (−/−) total cell extracts. Protein complexes were affinity-purified with glutathione-Sepharose and analyzed by SDS-PAGE and Western blotting with anti-CBP and anti-GST, to ensure that similar levels of fusion proteins were present in all cases. In the Input lane, a sample corresponding to 5% of the total cell extracts used for the assay was loaded. Autoradiograms were scanned and the average of CBP bound with respect to the value obtained without CKI is shown below the western blot (average +/− S.D. of three experiments). (B) MEFs PS (−/−) were transfected with 7 µg of GFP or E-cadh-CTF2-GFP, to simulate the CTF2 fragment present in MEF PS1/PS2 (+/+). After 24 h expression, cells were treated with the proteosome inhibitor MG132 at 30 µM for 4 h. RIPA buffer was used to obtain cytosolic cell fraction. For the IP, 500 µg of the cytosolic fraction were incubated with 2.5 µg of anti CBP antibody to a final lysate concentration of 1 µg/µl. 30 µg of cytosolic cell lysates were loaded for inputs.</p

p120-catenin prevents the interaction of pPS1 with E-cadherin, the generation of E-cadherin-CTF2 fragment and the inhibition of β-catenin·Tcf-4 transcriptional activity.

<p>(A) MEF PS (−/−) cells were transfected with npPS1 or pPS1: pcDNA3.1-<i>Myc/His</i>-PS1 (npPS1) or pcDNA3.1-PS1 (pPS1). After 48 h total cell extracts were prepared and incubated with 15 pmols of GST-E-cadh/CTF2 or GST as a control; cell extracts from MEFs PS (+/+) were also incubated. Protein complexes were purified with glutathione-Sepharose and associated PS1 was analyzed by Western blotting with a specific antibody against PS1 (C-terminus). Blots were re-analyzed with anti-GST to ensure that similar levels of fusion protein forms were present in all samples. In the Input lane, a sample containing 5% of the total cell extracts used for the assay was loaded. (B) 12 pmol of GST-E-cadh-CTF2 or GST as a control were pre-incubated with 20 pmols of p120-catenin and pulldown assays performed as above with 1 mg of MEF PS (+/+) total cell extracts. (C) MEF PS (−/−) cells were cotransfected with 2.5 µg of pECFPN1-E-cadherin and pcDNA3.1-PS1 (pPS1) or pcDNA3.1-p120-catenin. 50 µg of total cell extracts were analyzed by SDS-PAGE and Western blot with the indicated antibodies. At the right, the ectopic E-cadherin levels of three different experiments were quantified and represented with respect to control (no additions). Values are the average +/− S.D of these experiments. (D) MEF PS1(−/−) cells were cotransfected with pcDNA3.1-PS1 (pPS1), pcDNA3.1-<i>Myc/His</i>-PS1 (npPS1), pcEGFPC1-E-cadh-CTF2, pcDNA3.1-p120-catenin or empty vector (150 ng), TOP-FLASH (50 ng) and pTK-<i>Renilla</i> (10 ng) luciferase plasmids. MEF PS1(+/+) cells were transfected with pcDNA3.1-p120-catenin or empty vector (150 ng). Relative luciferase activity was determined with a dual luciferase reporter assay system 48 h after transfection, and the result was normalized using the <i>Renilla</i> luciferase activity for each sample. Percentage activity was calculated by comparing levels of luciferase activity with levels after transfection of the empty plasmid alone. Values are the average +/− S.D. of three-four experiments performed in triplicate. −, absent; +, present. (*) indicates p<0.05; (**), p<0.01; in the rest of the comparisons, the p value is shown. NS, not significant.</p

Processed and non-processed PS1 block β-catenin·Tcf-4-dependent transcription differently.

<p>SW-480 (A,) and MEF PS (−/−) cells (C) were transfected with 5 µg of pcDNA3 plasmid containing either <i>Myc/His</i>-tagged PS1 (npPS1), wild-type PS1 (pPS1), the indicated mutants, or empty vector as a control. After 48 hours (A) or at the indicated times (C), cell extracts were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004080#s2" target="_blank"><i>Materials and Methods</i></a>. 50 µg of untransfected or transfected total cell extracts were analyzed by SDS-PAGE and Western blot with antibodies anti-PS1 (amino acids 1–65) and anti-β-actin as a control (A and C). In panels B and D cells SW-480 and MEFs were cotransfected with 150 (+, in B) or 300 ng (++, in B; and D) of pcDNA-3 containing npPS1, pPS1, or, the indicated mutants, plus TOP-FLASH (50 ng) and pTK-<i>Renilla</i> (10 ng) luciferase plasmids. Relative luciferase activity was determined with a dual luciferase reporter assay system 48 hours after transfection (B) or at the indicated times (D), and the result was normalized using the <i>Renilla</i> luciferase activity for each sample. Percentage activity was calculated by comparing levels of luciferase activity with levels after transfection of the empty plasmid alone. 5 µM γ-secretase inhibitor L-685,458 (Calbiochem) was added to the medium for the last 24 h (panels B and D). Values are the average +/− S.D. of three-four experiments performed in triplicate. One asterisk (*) indicates p<0.05; two asterisks (**) p<0.01; in the rest of the comparisons, the value of p is presented.</p