MUC1 downregulation by shRNA.

<p>(<b>A</b>) MUC1 detection by immunofluorescence with HMFG-1 antibody in MKN45-C1 and MKN45-C2 and MKN45-SC control; (<b>B</b>) MUC1 protein detection by western-blot with MUC1-Ab5 antibody of total protein extracts from MKN45-C1 and MKN45-C2 and MKN45-SC control; (<b>C</b>) Quantification of MUC1 RNA in MKN45-C1 and MKN45-C2 and MKN45-SC control by real-time PCR. MUC1 expression was corrected to the house-keeping gene 18S and normalized to the data obtained with the scrambled control. <i>*P</i><0.01.</p

Study of the tumorigenicity of MKN45 gastric carcinoma cells <i>in vivo</i>.

<p>Tumor growth curves. 1×10⁵ cells were subcutaneously injected in mice at day 0. The curves show tumor growth until day 21, the day on which all mice were sacrificed.* P<0.05, when compared to the MKN45-SC control cell line.</p

Quantification of cell proliferation by MTT assay.

<p>Quantification of metabolically active cells by MTT assay in MKN45-C1 and MKN45-C2 clones and MKN45-SC control at 96h in culture. Data from 24 hours was used to set time zero and results were normalized to the data obtained with the scrambled control.<i>*P</i><0.01.</p

Quantification of apoptotic cells by TUNEL assay.

<p>Apoptosis of MKN45-C1 and MKN45-C2 and scramble control (SC) were evaluated at 96h in culture by the TUNEL assay. Results were normalized to the data obtained with the scrambled control. <i>*P</i><0.01.</p

Quantification of cell-cell aggregation.

<p>(<b>A</b>)Quantification of the cell-cell aggregation index in MKN45-C1 and MKN45-C2 and MKN45-SC control. The cell-cell aggregation index was assessed by the observed decrease in the number of isolated cells over time, and normalized to the data obtained with the scrambled control. <i>*P</i><0.01; (<b>B</b>) Images of the aggregates formed after 1 and 2 hours of constant stirring. First column shows isolated cells at time 0h (20x magnification) and second and third columns show aggregates formed after 1h and 2h of incubation (40x magnification), in MKN45-C1 and MKN45-C2 and MKN45-SC control.</p

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

<div><p>Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.</p> </div

hPaf1 is ubiquitinylated and degraded at the metaphase/anaphase transition.

<p>Panc1 cells were grown at low density on cover slips for 24 h, and arrested at G1/S (T0) and G2/M (N0) boundaries by double thymidine and thymidine/nocodazole blocks, respectively. Acetone/methanol (1∶1)-fixed Panc1 cells were labeled using a FITC-conjugated anti-hPaf1 monoclonal antibody and counterstained by propidium iodide. Up to 10 fields were observed by confocal microscopy for cells after 12 h of double thymidine treatment release (T+12) and compared with 10 fields of thymidine/nocodazole treated cells just before release (N0). Representative pictures are presented in A). The black arrows indicate the inter-chromatine accumulation of hPaf1 in the prophase for the cells in N0; accumulation was not detected at T+12. B) Proteins from nuclear extracts performed at T0, T+8, N0, and S0 (shake off cells in N0) were immunoprecipitated using the rabbit polyclonal antibody anti-ubiquitin (Calbiochem), and immunoblotted with anti-hPaf1 antibody. An aliquot of the nuclear protein extracts was loaded as a control for hPaf1 expression (input), and immunoprecipitation was carried out using an isotype IgG control antibody as a negative control. C) hPaf1 degradation by the proteasome was confirmed using the proteasome inhibitor MG132. After double thymidine block, the cells were released either in ethanol control or MG132 (20 µM) for 12 h. Protein lysates were imunoprecipitated using the rabbit polyclonal antibody anti-ubiquitin (Calbiochem) and immunoblotted with anti-hPaf1 antibody. D) Panc1, CD18/HPAF, and human fibroblast cells were arrested at the G1/S boundary and released for 12 h to reach a maximum of mitosis. The cells were analyzed for hPaf1 expression and sub cellular localization by confocal. In the prophase, hPaf1 was found aligned on filament-like structures (D3) to be concentrated at the pole in the metaphase (D1, D2: black arrow). The Z-section (D4, D5, and D6) revealed that in the metaphase, hPaf1 formed a crown at both poles of the cells, potentially surrounding the centrosomes (white arrow). E) CD18/HPAF cells were arrested at the G1/S boundary and released for 12 h to reach a maximum of mitosis. The cells were analyzed for hPaf1 and tubulin expression and subcellular localization by confocal. In metaphase, hPaf1 co-localized with tubulin, confirming the centrosomal localization of hPaf1.</p

Quantitative analysis of miR-200c expression in human pancreatic cancer cell lines and ectopic expression of miR-200c in S2.028 and T3M-4 cells.

<p>(A) The expression of miR-200c in seven pancreatic cancer cells were determined by Real-time PCR. Each sample was run in quadruplicate and error bars represent SD. S2.028 (B) and T3M-4 cells (C) stably expressing the primary transcript of miR-200c were evaluated for miR-200c expression by Real-time PCR. Each measurement was carried out in triplicate. These values were normalized with internal control U6 rRNA. The fold increase in transcript levels over vector control is expressed as Mean ± S.D. The p value was determined by using the Student’s t-test. Differences with a p value < 0.05 were considered statistically significant.</p

Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution

<div><p>The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation–pain and swelling–by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process <i>in vivo</i>. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary <i>in vivo</i> approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75^NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities <i>in vitro</i>. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis <i>in vivo</i>. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.</p></div

Prediction of miR-200c interaction sites in MUC4 and MUC16 genes.

<p>Possible miR-200c targeting regions in MUC4 and MUC16 were identified by using the RegRNA MicroRNA target prediction web server (<a href="http://regrna.mbc.nctu.edu.tw/index1.php" target="_blank"><u>http://regrna.mbc.nctu.edu.tw/index1.php</u></a>). A, RegRNA miRNA target prediction shows that miR-200c binds between base pairs 820-842 in the first exon of MUC4. B, In MUC16 mRNA, the miR-200c is predicted to bind nine different exons including E1, E3, E19, E39, E44, E49, E54, E64 and E73. The numbers indicate the region of mRNAs that interact with miR-200c.</p