The EMT state.Furthermore, we wanted to see if these cells

The EMT state.Furthermore, we wanted to see if these cells also showed different expression levels of HER family members and proteins of their signaling pathways. We observed that the levels of all HER family members and their downstream signaling partners were downregulated in all resistant cancer cell lines (Figs. 5B and S4). A suppression of downstream signaling was similarly seen in the breast and pancreatic resistant cell lines, and the same expression pattern was also observed in other colon and lung resistant cell lines, highlighting the relevance of this phenomena.Modulation of HER3 Affects Cancer Cell Line Sensitivity to ElisidepsinBased on previous studies from our group and others demonstrating that Title Loaded From File elisidepsin downregulates the HER3 receptor tyrosine kinase and that high expression of HER3 is prevalent in a broad number of different tumor cells, we investigated if modulation of protein expression levels of the HER3 receptorEMT and HER3 Predicts Elisidepsin SensitivityFigure 2. Expression of EMT markers associated with elisidepsin sensitivity in breast cancer cell lines. Protein expression levels of different EMT markers were evaluated by immunocytochemistry (A), western blot (B) and IHC (C). A) Immunocytochemistry of two epithelial (Ecadherin and b-catenin) and four mesenchymal markers (vimentin, Slug, Snail and Twist). Magnification 100x. B) E-cadherin, b-catenin, Slug, Snail, Twist, vimentin and b-actin (loading control) were detected by western blot analysis using 50 mg of total protein. C) Basal levels of E-cadherin, bcatenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate. doi:10.1371/journal.pone.0053645.gaffects sensitivity to elisidepsin in a panel of tumor cell lines with variable expression of this receptor. To examine this experimentally, we utilized a shRNA construct to stably reduce HER3 expression in a panel of cell lines (Fig. 6).Stable clones of HER3 shRNA and LUC shRNA (control) vectortransfected cells were selected and examined for expression of HER3. As expected, levels of HER3 were significantly reduced in HER3-transfected cells, but not in those containing the pLKOEMT and HER3 Predicts Elisidepsin SensitivityFigure 3. Expression of EMT markers associated with elisidepsin sensitivity in pancreatic cancer cell lines. E-cadherin, b-catenin, vimentin, Slug, Snail and Twist basal expression levels were evaluated by immunocytochemistry (A) and western blot (50 mg of protein/lane) (B). Magnification 100x. Membranes were stripped and reprobed with anti-b-actin as an internal control. C) E-cadherin, b-catenin and vimentin protein expression levels were evaluated by IHC. Magnification 20x. These analyses were performed in duplicate. doi:10.1371/journal.pone.0053645.gLUC shRNA vector alone, indicating that the decrease in HER3 was not due to non-specific effects of introducing shRNA into the cells. Next, cell viability assays were performed to analyze elisidepsin sensitivity in the generated cells. Figure 6 shows that cells that have reduced levels of HER3 due to Title Loaded From File shRNA-mediated knockdown of its expression showed loss of sensitivity to elisidepsin treatment in comparison to control cell lines.To investigate whether ectopic HER3 expression affects the elisidepsin sensitivity of low HER3-expressing cells, the levels of HER3 were increased by transfecting cells with a cDNA encoding HER3, which resulted in increased sensitivity of the cells to elisidepsin. In com.The EMT state.Furthermore, we wanted to see if these cells also showed different expression levels of HER family members and proteins of their signaling pathways. We observed that the levels of all HER family members and their downstream signaling partners were downregulated in all resistant cancer cell lines (Figs. 5B and S4). A suppression of downstream signaling was similarly seen in the breast and pancreatic resistant cell lines, and the same expression pattern was also observed in other colon and lung resistant cell lines, highlighting the relevance of this phenomena.Modulation of HER3 Affects Cancer Cell Line Sensitivity to ElisidepsinBased on previous studies from our group and others demonstrating that elisidepsin downregulates the HER3 receptor tyrosine kinase and that high expression of HER3 is prevalent in a broad number of different tumor cells, we investigated if modulation of protein expression levels of the HER3 receptorEMT and HER3 Predicts Elisidepsin SensitivityFigure 2. Expression of EMT markers associated with elisidepsin sensitivity in breast cancer cell lines. Protein expression levels of different EMT markers were evaluated by immunocytochemistry (A), western blot (B) and IHC (C). A) Immunocytochemistry of two epithelial (Ecadherin and b-catenin) and four mesenchymal markers (vimentin, Slug, Snail and Twist). Magnification 100x. B) E-cadherin, b-catenin, Slug, Snail, Twist, vimentin and b-actin (loading control) were detected by western blot analysis using 50 mg of total protein. C) Basal levels of E-cadherin, bcatenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate. doi:10.1371/journal.pone.0053645.gaffects sensitivity to elisidepsin in a panel of tumor cell lines with variable expression of this receptor. To examine this experimentally, we utilized a shRNA construct to stably reduce HER3 expression in a panel of cell lines (Fig. 6).Stable clones of HER3 shRNA and LUC shRNA (control) vectortransfected cells were selected and examined for expression of HER3. As expected, levels of HER3 were significantly reduced in HER3-transfected cells, but not in those containing the pLKOEMT and HER3 Predicts Elisidepsin SensitivityFigure 3. Expression of EMT markers associated with elisidepsin sensitivity in pancreatic cancer cell lines. E-cadherin, b-catenin, vimentin, Slug, Snail and Twist basal expression levels were evaluated by immunocytochemistry (A) and western blot (50 mg of protein/lane) (B). Magnification 100x. Membranes were stripped and reprobed with anti-b-actin as an internal control. C) E-cadherin, b-catenin and vimentin protein expression levels were evaluated by IHC. Magnification 20x. These analyses were performed in duplicate. doi:10.1371/journal.pone.0053645.gLUC shRNA vector alone, indicating that the decrease in HER3 was not due to non-specific effects of introducing shRNA into the cells. Next, cell viability assays were performed to analyze elisidepsin sensitivity in the generated cells. Figure 6 shows that cells that have reduced levels of HER3 due to shRNA-mediated knockdown of its expression showed loss of sensitivity to elisidepsin treatment in comparison to control cell lines.To investigate whether ectopic HER3 expression affects the elisidepsin sensitivity of low HER3-expressing cells, the levels of HER3 were increased by transfecting cells with a cDNA encoding HER3, which resulted in increased sensitivity of the cells to elisidepsin. In com.