Of Twist-1 and vimentin were less sensitive to the drug (Figs. 2A ). In contrast, elisidepsin-sensitive pancreatic carcinoma cell lines MedChemExpress 223488-57-1 expressed E-cadherin and b-catenin, whereas the less sensitive cells expressed Slug. Lastly, Snail, Twist-1 and vimentin expression was found in sensitive and insensitive cell lines alike (Figs. 3A ). To summarize, E-cadherin protein was significantly expressed in the sensitive cell lines independently of their tumoral origin (Mann-Whitney test: p = 0.0364; Fig. S2), and vimentin was significantly expressed in the less sensitive ones (Mann-Whitney test: p = 0.0364). On the other hand, Twist-1 and Snail proteins were found in all less sensitive cell lines (Mann Whitney test: p = 0.0636 and p = 0.1000, respectively), with the exception of two sensitive cell lines that were positive for vimentin expression (CFPAC and AsPC-1), one sensitive cell line that was positive for Twist-1 expression (CFPAC) and another one that was positive for Snail expression (SKBR3).HER3 Expression Levels Correlate with Elisidepsin Cell SensitivityThe primary mechanisms of action of elisidepsin remain to be elucidated but we and other groups have found that after 4 h treatment with 1 mM elisidepsin, HER3 receptor levels are downregulated in a panel of different cell lines, including lung, breast, melanoma and colon 
carcinomas [10,11]. To determine if HER3 protein expression levels correlate with the sensitivity of the cell lines to elisidepsin, we performed IHC (Fig. 4A) and western blot analysis (Fig. 4B) in all cell lines. Cell lines that were less sensitive to elisidepsin had little to no HER3 while sensitive cell lines expressed significantly increased levels of this protein (MannWhitney test: p = 0.0091; Fig. S3). In addition, others members of the HER family were checked by western blot (Fig. 4B) but no correlations with elisidepsin sensitivity were found with HER1, HER2 and HER4 (Mann-Whitney test: p = 0.7273, p = 0.5182 and p = 0.8909, respectively).Acquired Resistance to Elisidepsin Induces an EMT PhenotypeThree elisidepsin-resistant cancer cell lines [one breast (MCF-7) and two pancreatic (HPAC, AsPC-1)] were generated by continuous exposure to increasing concentrations of the drug (see Material and Methods). Cancer cell lines were exposed to elisidepsin at a starting concentration of its IC50. Elisidepsin concentration was increased every week until cells became resistant to the drug, after approximately 12 months in the case of MCF-7, and after approximately 4 months in the case of HPAC and AsPC-1. The morphology of the resistant cancer cell lines was modified after continuous exposure to the drug when compared to that of the parental cell lines (data not shown). Our hypothesis was that the loss of epithelial ��-Sitosterol ��-D-glucoside markers observed in our panel of cancer cell lines could be responsible for the resistance to elisidepsinCorrelation between EMT Markers and Elisidepsin Cell SensitivityIn order to evaluate EMT protein expression levels and correlate them with the sensitivity of the cell lines to elisidepsin, we performed different analyses using western blot, immunofluorescence and immunohistochemistry (IHC) in a panel of 12 cellEMT and HER3 Predicts Elisidepsin SensitivityFigure 1. Elisidepsin sensitivity. A) Elisidepsin IC50s were determined in a panel of breast (left) and pancreatic (right) cancer cell lines using a crystal violet assay. Cells were exposed to elisidepsin for 72 h. Results are shown as the mean 6 SD of at lea.Of Twist-1 and vimentin were less sensitive to the drug (Figs. 2A ). In contrast, elisidepsin-sensitive pancreatic carcinoma cell lines expressed E-cadherin and b-catenin, whereas the less sensitive cells expressed Slug. Lastly, Snail, Twist-1 and vimentin expression was found in sensitive and insensitive cell lines alike (Figs. 3A ). To summarize, E-cadherin protein was significantly expressed in the sensitive cell lines independently of their tumoral origin (Mann-Whitney test: p = 0.0364; Fig. S2), and vimentin was significantly expressed in the less sensitive ones (Mann-Whitney test: p = 0.0364). On the other hand, Twist-1 and Snail proteins were found in all less sensitive cell lines (Mann Whitney test: p = 0.0636 and p = 0.1000, respectively), with the exception of two sensitive cell lines that were positive for vimentin expression (CFPAC and AsPC-1), one sensitive cell line that was positive for Twist-1 expression (CFPAC) and another one that was positive for Snail expression (SKBR3).HER3 Expression Levels Correlate with Elisidepsin Cell SensitivityThe primary mechanisms of action of elisidepsin remain to be elucidated but we and other groups have found that after 4 h treatment with 1 mM elisidepsin, HER3 receptor levels are downregulated in a panel of different cell lines, including lung, breast, melanoma and colon carcinomas [10,11]. To determine if HER3 protein expression levels correlate with the sensitivity of the cell lines to elisidepsin, we performed IHC (Fig. 4A) and western blot analysis (Fig. 4B) in all cell lines. Cell lines that were less sensitive to elisidepsin had little to no HER3 while sensitive cell lines expressed significantly increased levels of this protein (MannWhitney test: p = 0.0091; Fig. S3). In addition, others members of the HER family were checked by western blot (Fig. 4B) but no correlations with elisidepsin sensitivity were found with HER1, HER2 and HER4 (Mann-Whitney test: p = 0.7273, p = 0.5182 and p = 0.8909, respectively).Acquired Resistance to Elisidepsin Induces an EMT PhenotypeThree elisidepsin-resistant cancer cell lines [one breast (MCF-7) and two pancreatic (HPAC, AsPC-1)] were generated by continuous exposure to increasing concentrations of the drug (see Material and Methods). Cancer cell lines were exposed to elisidepsin at a starting concentration of its IC50. Elisidepsin concentration was increased every week until cells became resistant to the drug, after approximately 12 months in the 
case of MCF-7, and after approximately 4 months in the case of HPAC and AsPC-1. The morphology of the resistant cancer cell lines was modified after continuous exposure to the drug when compared to that of the parental cell lines (data not shown). Our hypothesis was that the loss of epithelial markers observed in our panel of cancer cell lines could be responsible for the resistance to elisidepsinCorrelation between EMT Markers and Elisidepsin Cell SensitivityIn order to evaluate EMT protein expression levels and correlate them with the sensitivity of the cell lines to elisidepsin, we performed different analyses using western blot, immunofluorescence and immunohistochemistry (IHC) in a panel of 12 cellEMT and HER3 Predicts Elisidepsin SensitivityFigure 1. Elisidepsin sensitivity. A) Elisidepsin IC50s were determined in a panel of breast (left) and pancreatic (right) cancer cell lines using a crystal violet assay. Cells were exposed to elisidepsin for 72 h. Results are shown as the mean 6 SD of at lea.
