SnoN expression is reduced in pancreatic adenocarcinoma samples.

<p><b>A,</b> Representative SnoN expression in the normal pancreas and in pancreatic adenocarcinoma of varying grades at 20X (top) or 40X (bottom) magnifications. E: epithelium; S: stroma. Green: SnoN; blue, DAPI. <b>B,</b> SnoN staining in normal and pancreatic tumor epithelial cells was quantified using Image J, and the numbers were plotted in the box plot, which includes normal samples (n = 5, mean = 3.08) and pancreatic tumor samples of grade I (n = 21, mean = 1.89), grade II (n = 59, mean = 1.59), and grade III (n = 8, mean = 2.09). SnoN expression in tumor samples was weaker than that in normal pancreatic samples (p = 0.0855 for grade I, p = 0.0125 for II, and p = 0.0518 for III). No significant difference was observed in SnoN epithelial staining between the pancreatic tumor samples. <b>C,</b> SnoN staining in normal (n = 2, mean = 1.87) and tumor stromal samples of grade I (n = 20, mean = 2.10), II (n = 55, mean = 1.70), and III (n = 8, mean = 1.57). There is no statistically significant difference between tumor and normal stroma samples.</p

SnoN expression in esophageal adenocarcinoma.

<p><b>A,</b> Representative SnoN staining of esophageal cancer of various grades at 20X (top) or 40X (bottom) magnifications. Two grade III samples representing different levels of SnoN expression were shown. E: epithelium; S: stroma. Green: SnoN; blue, DAPI. <b>B,</b> SnoN staining in normal and tumor epithelial cells was quantified using the Image J software and the numbers were plotted in the box plot, which includes normal samples (n = 36, mean intensity = 1.13) and esophageal tumor samples of grade I (n = 8, mean = 0.07), II (n = 19, mean = 0.71), and III (n = 11, mean = 1.26). Statistical analysis comparing the normal controls to each tumor grade showed that the epithelial SnoN levels in esophageal adenocarcinoma are significantly weaker (grade I: p = 0.0002) or similar (grade II: p = 0.1425 and grade III: p = 0.3349) to that in the control normal samples. The increase in epithelial SnoN expression in grade III compared to grade I was statistically significant (p = 0.0013)<b>. </b><b>C,</b> Quantification of SnoN stromal staining in normal samples (n = 27, mean = 1.69) and esophageal tumor samples of grade I (n = 5, mean = 0.23), II (n = 19, mean = 1.09), and III (n = 11, mean = 1.78). The statistical analysis comparing the normal controls to each esophageal tumor grade is as follow: p = 0.0023 for grade I, p = 0.8565 for II, and p = 0.1132 for grade III. The increase in stromal SnoN expression in grade II (p = 0.0287) and grade III (p = 0.0068) tumors compared to grade I tumor stroma was statistically significant.</p

Elevated SnoN expression correlates with inactivation of p53 in human cancer cell lines but not in primary tumor tissues.

<p><b>A,</b> 914 cancer cell lines from the Novartis CLE were classified based on their p53 gene status (lost or wild-type) as shown in the X-axis and their correlation with the copy numbers of SnoN as indicated in the Y-axis. A significant enrichment of SnoN amplification events in p53 mutant or deleted cell lines was identified (p = 7.25E-009). <b>B,</b> Cell lines from the CLE were divided into 18 different tissue lineages as depicted by various colors, and the correlation between the frequency of TP53 mutation (X-axis) and frequency of SnoN amplification (Y-axis) was determined to be highly significant with a Pearson’s correlation coefficient of 0.7. <b>C,</b> Representative p53 immunohistochemical stain in normal ovarian tissue and ovarian adenocarcinoma of grade I, grade II, and grade III (Original magnification ×20). <b>D,</b> Box plot depicting the intensity of epithelial SnoN expression (Y-axis) and p53 protein levels (as marked from 0 to 5, 0 being the lowest level in normal tissues and 5 being the highest). No significant correlation between the status of SnoN protein level and p53 inactivation was noted as measured by the Kruskal-Wallis test (p = 0.817).</p

SnoN is expressed in normal mammalian tissues. A,

<p>SnoN expression in the normal esophagus, including the suprabasal differentiated squamous epithelial cells, the lamina propria (stroma and connective tissue), and muscularis mucosa (smooth muscle). E: epithelial cells; F: fibroblasts; B.V; blood vessel. Negative control: tissue stained with conjugated secondary antibody alone and without primary antibody. Peptide control: tissue stained with the SnoN peptide competition control. Green: SnoN; blue, DAPI. <b>B,</b> Representative SnoN expression in the normal ovarian tissue. E: follicle epithelial cells; S: stroma. The left panel is DAPI stain alone (blue), the middle panel is SnoN stain alone (green), and the right panel is SnoN (green) plus DAPI (blue) stains. Same is true for figure panels in C-D. <b>C,</b> Representative SnoN expression in the normal pancreas. E: acinar epithelial cells; S:stromal cells of the lobular connective tissue septa. <b>D,</b> Representative SnoN expression in the normal breast. E: epithelial cells of ducts and lobuli; S: stroma.</p

POSTN promotes a mesenchymal phenotype in MCF-10A and MCF-7 cells.

<p><b>A.</b> POSTN-overexpressing cells exhibit a mesenchymal-like morphology. <b>B.</b> POSTN promotes cell invasion of human mammary epithelial cells and BCCs as detected by a matrigel-coated transwell invasion assay. <b>C, D.</b> Immunofluorescence analysis revealed that the mesenchymal markers N-cadherin, fibronectin, vimentin and α-SMA in POSTN-expressing cells were increased while the epithelial marker E-cadherin was decreased. <b>E, F.</b> POSTN-expressing cells show increased levels of N-cadherin, fibrnectin, vimentin and α-SMA and decreased E-cadherin. Expression of epithelial and mesenchymal markers was analysed by western blotting.</p

Model of role of POSTN in mammary epithelial neoplasia and metastasis.

<p>POSTN might confer mammary epithelial cells and BCCs with stem cell-like traits and a mesenchymal phenotype, as well as the multipotent potentials of MSCs to promote tumorigenesis and metastasis.</p

POSTN induces adipogenic and chondrogenic differentiation.

<p><b>A.</b> Following adipogenic differentiation, MCF-10A/POSTN, MCF-7/POSTN cells and hMSCs stained positive with oil red O (top) and fluorescent LipidTox, which stains oil droplets (bottom). <b>B, C.</b> Real-time RT-PCR analysis for the expression of the adipocyte markers <i>PPARγ</i>and <i>ADFP</i> in MCF-10A and MCF-7 cells and their POSTN-overexpressing cells subjected to adipocyte differentiation for 21 days. The data are means ± SD. *P<0.05, **P<0.01. <b>D.</b> Chondrocytic nodules formed by MCF-10A/POSTN cells and hMSCs stained positive with alcian blue 8 GX (left panel). Immunohistochemistry was performed on chondrocyte sections using antibody against collagen II (right panel). MCF-10A/Vector cells, MCF-7/Vector and MCF-7/POSTN cells did not form any chondrocytic nodules under identical conditions.</p

POSTN induces osteoblastic and myogenic differentiation.

<p><b>A, B.</b> Following culture in osteoblastic differentiation media for 21 days, MCF-10A/POSTN, MCF-7/POSTN cells and hMSCs were positive for alizarin red S and von Kossa staining. <b>C, D.</b> Relative levels of mRNAs encoding BSP and Runx2 in MCF-10A and MCF-7 cells expressing the vector or POSTN were determined by real-time RT-PCR. <i>Cyclophilin G</i> mRNA was used to normalize the variability in template loading. The data are the means ± SD. *P<0.05, **P<0.01. <b>E.</b> Following myogenic differentiation for 4 weeks, MCF-10A/POSTN cells stained positive for CD56. MCF-7/POSTN cells died under the same myogenic differentiation condition and did not differentiate into myogenic lineages (data not shown). <b>F.</b> Real-time RT-PCR analysis of <i>MyoG</i> and <i>Pax3</i> showing the expression of myogenic markers. The data are the means ± SD. *P<0.05.</p

POSTN-overexpressing cells accelerates the tumor growth and metastatic properties of BCCs.

<p><b>A.</b> 2.5×10⁶ MCF-7/Vector or MCF-7/POSTN cells were injected subcutaneously into 5- to 6-week-old female Balb/c nude mice (n = 4 mice per group). Mice were sacrificed 30 days after injection and examined for the growth of subcutaneous tumors. **P<0.01. <b>B.</b> Tumor weight measurements of 1×10⁶ MDA-MB-231 cells subcutaneously injected into nude mice with 3×10⁶ MCF-10A/Vector cells or MCF-10A/POSTN cells. <b>C.</b> The tumors were measured once or twice per week. Circles, MDA-MB-231 cells; squares, MDA-MB-231 cells plus MCF-10A/Vector cells; triangles, MDA-MB-231 cells plus MCF-10A/POSTN cells; diamonds, MCF-10A/POSTN cells (n = 4 mice per group). **P<0.01. <b>D.</b> Mice subcutaneously implanted with 3×10⁶ MDA-MB-231 mixed with 9×10⁶ MCF-10A/Vector cells or with MCF-10A/POSTN cells (n = 3 mice per group). Twelve weeks after cell injection, the mice were euthanized and the lung metastasis indices for each tumor bearing mouse were determined. *P<0.05. <b>E.</b> Representative haematoxylin-and-eosin-stained sections of lungs of mice bearing the indicated tumors in (D). Scale bar = 500 µm.</p