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Time course of H2A.X phosphorylation after SAHA and Actinomycin D treatment. LA1-55n cells were treated (+) or not (-) with 0.1 nM of actinomycin D (ActD) in the presence (+) or in the absence (-) of 1 μ M SAHA. Indicated protein expression was determined by Western blot analysis at the indicated times after the treatment. (DOCX 710 kb

HER3 expression levels correlate with cell sensitivity to elisidepsin.

<p>A) Cell pellets were fixed in formalin, embedded in paraffin and a HER3 IHC was performed. Cell lines more sensitive to elisidepsin had significant HER3 levels. Magnification 40x. B) Basal expression levels of HER family members were analyzed by western blot; an association between HER3 expression and elisidepsin sensitivity was observed (Mann-Whitney test: p  = 0.0091; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053645#pone.0053645.s003" target="_blank">Fig. S3</a>). Cell lines less sensitive to elisidepsin (MDA-MB-231, PANC-1 and MiaPaCa-2) did not show significant HER3 protein levels, while PANC-1 and MiaPaCa-2 cell lines show levels of other HER family members. No correlation was observed with HER1, HER2 and HER4 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053645#pone.0053645.s003" target="_blank">Fig. S3</a>). These protein expression levels were analyzed in duplicate and 50 µg of protein of cell lysate were loaded in each lane.</p

Acquired resistance to elisidepsin induces an EMT phenotype.

<p>A) Cells were lysed, proteins were extracted and western blots were performed with equal amounts of cell lysate (50 µg protein). Expression of epithelial (E-cadherin, β-catenin, γ-catenin)- and mesenchymal (vimentin, Slug, Snail, Twist)-associated proteins differentiates between elisidepsin-sensitive and elisidepsin-resistant cell lines. β-actin was used as an internal control. These western blots were performed in triplicate. B) Expression levels of HER1, HER2, HER3, HER4, pAkt, and pMAPK were analyzed by western blot using 50 µg of protein cell lysate. The membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. C, control; R, resistance.</p

Loss of HER3 expression decreases the sensitivity to elisidepsin treatment.

<p>Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in SKBR3 (A), MCF-7 (B), MDA-MB-231 (C), MDA-MB-435 (D), BT474 (E), BxPC-3 (F), HPAC (G) and AsPC-1 (H) cells. HER3 expression was downregulated with shRNA (grey squares); LUC shRNA transfected cells were used as the control (black diamonds). Mean, SD, and IC₅₀ values are shown from three independent experiments. Cell viability was measured using a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 µg of protein to confirm their levels of HER3 expression.</p

Expression of EMT markers associated with elisidepsin sensitivity in breast cancer cell lines.

<p>Protein expression levels of different EMT markers were evaluated by immunocytochemistry (A), western blot (B) and IHC (C). A) Immunocytochemistry of two epithelial (E-cadherin and β-catenin) and four mesenchymal markers (vimentin, Slug, Snail and Twist). Magnification 100x. B) E-cadherin, β-catenin, Slug, Snail, Twist, vimentin and β-actin (loading control) were detected by western blot analysis using 50 µg of total protein. C) Basal levels of E-cadherin, β-catenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate.</p

Elisidepsin sensitivity.

<p>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 ± SD of at least three independent experiments. B) Cell proliferation in parental and subtoxic elisidepsin-treated cells. Cumulative numbers of cell divisions [shown as population doubling level (PDL)] are shown for MCF-7 and MiaPaCa-2 cells until passage 5. Proliferation of MCF-7 (IC₅₀∶0.4 µM) and MiaPaCa-2 (IC₅₀∶14 µM) cells was suppressed when elisidepsin was added to the culture at subtoxic doses (0.2 and 1 µM, respectively). The number of MiaPaCa-2 and MCF-7 seeded cells were 1.25×10⁵ and 1.4×10⁵, respectively. Each growth curve was performed at least twice with similar results, SDs are shown, and each time point was performed in duplicate. P, passage.</p

Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).

<p>Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).</p

Percentage of mice developing adenomas and adenocarcinomas.

<p>Hematoxylin and eosin staining of histological sections showing lung hyperplasia in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ (<b>A</b>, <b>B</b>) and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo mice (<b>J</b>, <b>K</b>). Higher magnification is showed in (<b>B</b>, <b>K</b>). Papillary adenomas developed in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ (<b>C</b>, <b>D</b>) and mixed papillary and solid adenomas developed in CMV-<i>Cre</i>^T/+; <i>K-ras</i>^+/LSLG12Vgeo (<b>L</b>, <b>M</b>). Note <b>C</b> and <b>L</b> tumors in higher magnification (<b>D</b>, <b>M</b>). Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ adenocarcinoma (<b>E</b>) showing papillary (<b>F</b>) and solid (<b>G</b>) regions. Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ adenocarcinoma showing intra bronchiolar tumor growth (*) (<b>H</b>). Higher magnification showing different cells populations in <b>H.</b> Atypical cells with nuclear hyperchromasia, and contour irregularities (*), cells showed enlarged nuclei displaying prominent nucleoli (**) and cells with hyperchromatic fusiform nuclei (arrows). CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenomas and adenocarcinomas (<b>N</b>). Detail of solid (<b>O</b>) and mucinous (<b>P</b>) tumors. Dashed-lined squares indicate magnified areas. Bars 800 µm (<b>A</b>, <b>C</b>, <b>D</b>, <b>J</b>, <b>L</b> and <b>N</b>), 500 µm (<b>E</b>), 200 µm (<b>K</b>, <b>M</b>, <b>O</b> and <b>P</b>) and 100 µm (<b>B</b>, <b>D</b>, <b>F</b>, <b>G</b> and <b>I</b>).</p