MUC1.CT directly interacts with transcriptional repressor ZEB1.

<p>A) Co-immunoprecipitation (Co-IP) was utilized to evaluate the interaction of the MUC1.CT and ZEB1 in S2.013.Neo and S2.013.MUC1 cells. Cell lysates were immunoprecipitated with an antibody against the MUC1.CT, and the subsequent western was blotted with an antibody to ZEB1. Non-immunoprecipitated lysates were used as a loading control, and steady state levels of ZEB1 were higher in S2.013.MUC1 cells compared to S2.013.Neo cells. B-C) Proximity ligation assay (PLA) was used to visualize MUC1.CT and ZEB1 interaction in S2.013.Neo and S2.013.MUC1 cells, B, and Panc1.Neo and Panc1.MUC1 cells, C (with representative compressed z-stack image for each). These data show quantitative levels of MUC1.CT and ZEB1 interactions, indicating that MUC1.CT and ZEB1 interact in both cell lines. In the S2.013.Neo and S2.013.MUC1 cell lines, the interaction was significantly higher in the MUC1 expressing cells, confirming the Co-IP in A. However, there was no significant difference in interaction in the Panc1.Neo and Panc1.MUC1 cells. (Student’s t-test: *** p < 0.0005).</p

MiR-200c and MUC1 levels in a panel of pancreatic cancer cell lines and tumor samples.

<p>Correlation between levels of MUC1 and miR-200c in a panel of pancreatic cancer cell lines and a panel of primary tumor sections and liver metastases. A) MiR-200c levels, indicated by Ct value, are the white bars and correspond to the left y-axis. MUC1 statuses of these cell lines, determined by western blot, are represented in the black bars and right y-axis. Levels of miR-200c and their corresponding MUC1 levels [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073306#B20" target="_blank">20</a>], on the left and right y-axis respectively were evaluated in primary pancreatic tissue (B) and liver metastases (C). MUC1 status was determined based on IHC staining from one section per patient; therefore standard deviation was not determined.</p

qRT-PCR confirmation of microRNA levels in cells and pancreatic cancer tissue.

<p>MicroRNA levels were evaluated in triplicate by qRT-PCR to confirm expression changes identified by microarray analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073306#pone-0073306-t001" target="_blank">Table 1</a>). MicroRNA levels were normalized to the U6 RNA control. A) MiRs-200c, -141, -192, -33b, -194, -215, and -376c expression levels were evaluated in S2.013.Neo compared to S2.013.MUC1 cells. B) MiRs-200c, -141, -192, -33b, -194, -215, and -376c expression levels were evaluated in Panc1.Neo and Panc1.MUC1 cells. C) Levels of miR-200c were evaluated in a panel of pancreatic cancer tissues. These data are represented by Ct value, where lower Ct values indicate higher levels of miR-200c, and each data point represents a different patient sample. Uninvolved tissue is non-cancerous tissue isolated from a cancer patient. (Student’s t-test: *** p < 0.0005, ** p < 0.005, * p < 0.05).</p

Chromatin Immunoprecipitation confirmation of MUC1.CT and ZEB1 occupancy of the miR-200c/141 promoter.

<p>Chromatin immunoprecipitation (ChIP) was utilized to confirm MUC1.CT and ZEB1 occupancy at the miR-200c/141 promoter region. A) Schematic representing the miR-200c/141 promoter region where the ChIP primer sets were designed (-480 to -210, and +552 to +675) and the known ZEB1 binding region (Grey bar). B) ChIP results indicate fold change of normalized relative levels of MUC1.CT occupancy at the ZEB1 binding region (based on qPCR) of the miR-200c/141 promoter in S2.013.MUC1 cells compared to S2.013.Neo cells. C) ChIP results indicate fold change of normalized relative levels of ZEB1 occupancy at the ZEB1 binding region (based on qPCR) of the miR-200c/141 promoter in S2.013.MUC1 cells compared to S2.013.Neo cells. D) Sequence of the MUC1 cytoplasmic tail, indicating three phosphorylated tyrosine motifs that were examined here. E-F) ChIP results indicate relative levels (based on qPCR and normalized to IgG control) of phospho-YHPM, -YVPP, and -YEKV MUC1 cytoplasmic tail at the ZEB1 binding region of miR-200c/141 in the S2.013.MUC1 cells, E, and Panc1.MUC1 cells, F. In both cell lines, only the phospho-YEKV form of MUC1.CT shows significant enrichment at the promoter. All ChIP data was normalized to antibody specific IgG control. (Student’s t-test: *** p < 0.0005, ** p < 0.005, * p< 0.05).</p

PLA analysis of MUC1.CT interaction with ZEB1 in mitotic cells.

<p>Proximity ligation assay of MUC1.CT with ZEB1 was analyzed in mitotic cells versus non-mitotic cells. A) PLA results from mitotic (circled) or non-mitotic cells in S2.013.Neo or S2.013.MUC1, B indicate a roughly 2-3-fold increase in MUC1.CT and ZEB1 interaction. In addition, this interaction occurs mainly in the cytoplasm, not in the nuclei. (*** p < 0.0005).</p