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

Upregulation of HER3 increases elisidepsin sensitivity.

<p>Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in PANC-1 (A), MiaPaCa-2 (B), MDA-MB-435 (C) and MDA-MB-231 (D) cells. Stable cell lines with an upregulation of HER3 expression (with the pIRES HER3) are shown with white circles while black diamonds are used for LUC-transfected control cells (with the pIRES-LUC). Mean, SD, and IC₅₀ values are shown from three independent experiments. Cell viability was measured by 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

Proliferation defect in <i>Rplp1</i>^CNSΔ mouse embryonic brains.

<p>(<b>A</b>) BrdU staining of control and <i>Rplp1</i>^CNSΔ E13.5 embryonic brain paraffin sections after BrdU pulse-labeling for 1 h. Scale bars: 50 µm. N = 3. (<b>B</b>) Quantification of BrdU+ cells in the neocortex. (<b>C</b>) Ki67 staining in control and <i>Rplp1</i>^CNSΔ E13.5 embryonic brain paraffin sections. Scale bars: 50 µm. N = 3. (<b>D</b>) Quantification of Ki67+ cells in the neocortex. (<b>E</b>) Paraffin sections of control and <i>Rplp1</i>^CNSΔ E13.5 embryonic brains were stained for PH 3. Scale bars: 100 µm. N = 4. (<b>F</b>) Quantification of PH 3+ cells in the neocortex. (<b>G</b>) Paraffin sections of control and <i>Rplp1</i>^CNSΔ E15.5 embryonic brains were stained for Tuj1. Scale bars: 50 µm. N = 3. (<b>H</b>) Quantification of Tuj1+ cells in the neocortex. (<b>I</b>) Control and <i>Rplp1</i>^CNSΔ neurospheres formed <i>in vitro</i> after 7 days in culture. (<b>J</b>) Quantification of neurospheres per mL. Scale bars: 50 µm. N = 3. VZ: ventricular zone. SVZ: subventricular zone. IZ: intermediate zone. CP: cortical plate. Error bars: SEM.</p

RPLP1, a Crucial Ribosomal Protein for Embryonic Development of the Nervous System

<div><p>Ribosomal proteins are pivotal to development and tissue homeostasis. RP Large P1 (<i>Rplp1</i>) overexpression is associated with tumorigenesis. However, the physiological function of <i>Rplp1</i> in mammalian development remains unknown. In this study, we disrupted <i>Rplp1</i> in the mouse germline and central nervous system (<i>Rplp1^CNS</i>^Δ). <i>Rplp1</i> heterozygosity caused body size reductions, male infertility, systemic abnormalities in various tissues and a high frequency of early postnatal death. <i>Rplp1^CNS</i>^Δ newborn mice exhibited perinatal lethality and brain atrophy with size reductions of the neocortex, midbrain and ganglionic eminence. The Rplp1 knockout neocortex exhibited progenitor cell proliferation arrest and apoptosis due to the dysregulation of key cell cycle and apoptosis regulators (cyclin A, cyclin E, p21^CIP1, p27^KIP1, p53). Similarly, <i>Rplp1</i> deletion in pMEFs led to proliferation arrest and premature senescence. Importantly, <i>Rplp1</i> deletion in primary mouse embryonic fibroblasts did not alter global protein synthesis, but did change the expression patterns of specific protein subsets involved in protein folding and the unfolded protein response, cell death, protein transport and signal transduction, among others. Altogether, we demonstrated that the translation “fine-tuning” exerted by Rplp1 is essential for embryonic and brain development and for proper cell proliferation.</p></div

De novo protein synthesis is normal in <i>Rplp1</i>^iΔ MEFs, but the protein expression pattern is altered.

<p>(<b>A</b>) Western blot analysis of newly synthesized proteins in <i>Rplp1ⁱ</i>^Δ MEFs. Cycloheximide (CHX)-treated MEFs were used as a negative control. (<b>B</b>) Densitometric quantification of the newly synthesized proteins in <i>Rplp1</i>^iΔ MEFs. Two control and 2 <i>Rplp1ⁱ</i>^Δ MEF cell lines were used. Error bars: SEM. (<b>C</b>) Percentages of upregulated and downregulated proteins involved in different biological processes in <i>Rplp1^iΔ</i>pMEFs. The classification was performed according to gene ontology (<a href="http://www.geneontology.org" target="_blank">www.geneontology.org</a>). (<b>D</b>) Associated alterations in the levels of proteins that are dysregulated by a factor of 1.5 or more in <b><i>Rplp1ⁱ</i></b>^Δ<b>pMEFs</b> cells. Representative photographs of the 2D gel are shown. Only portions of the 2D gel images corresponding to the indicated proteins are shown.</p

MDA-MB-231 and HaCaT cells show increased resistance to stress after overexpression of phosphomimetic eIF4E.

<p>A, MDA-MB-231 and HaCaT cells were subjected to either arsenite (NaAsO₂), nutrient starvation, or cisplatin (CDDP) treatment, and cell viability was measured by an MTT assay after 24, 48, and 72 hours. In all cases, eIF4E-S209D significantly increased cell viability. B, Apoptotic activity measured by a caspase-3/-7 luminescence assay. Significant activation of caspase-3/-7 activity was observed following arsenite treatment in eIF4E-S209A– and GFP–expressing cells, which was completely prevented by eIF4E-S209D. * = P<0.05, ** P<0.01 and *** = P<0.001 compared to control, n = 3.</p