PyLT-induced Necdin expression is p53-independent.

<p>Necdin is induced following activation of p53. (A) Dose response treatment with nutlin-3 increased Necdin protein level in NIH (left) and NIHLT (right). (B) Genotoxic stress induced by Actinomycin D and Camptothecin also stimulated Necdin protein expression. (C) In NIHLT cells, Necdin expression is not dependent on p53 activity as assessed by p53 inhibition. Mean of relative expression of Necdin, p21, p53 and GAPDH in NIHLT cells with or without the p53 inhibitor GSE22. Expression was measured by Q-PCR in three independent samples from each group. Expression is relative to actin (**<i>P</i><0.001, t-test).</p

Necdin as a relevant candidate.

<p>(A) Validation of the microarray identified Necdin gene by Northern blot analysis on an independent extended clone set. Lanes 1 to 6 represent individual NIH3T3 sub-clones. Lanes 7 to 15 represent individual NIH3T3 clones transfected and selected to express PyLT. Clones were independent from those used in the microarray analysis. DIG-labeled probes were used and exposure times were adjusted. DOHX was used as a control. (B–C) Necdin protein level in (B) clones expressing variable level of PyLT or (C) additional heterogeneous populations of NIH3T3 cells stably transfected with PyLT (NIHLT) or empty vector (NIH). (D) Different mutant forms of PyLT protein expressed in NIH3T3 were used to determine the domain involved in Necdin modulation. Western blot shows protein expression levels. Representation of mutants utilized with Rb-binding and immortalization capacity as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031916#pone.0031916-Pilon1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031916#pone.0031916-Pilon2" target="_blank">[41]</a>.</p

Nutlin-3 induces a p53-dependent growth arrest in NIH3T3 cells that is bypassed by PyLT expression.

<p>(A–B) Flow cytometry analysis of NIH or NIHLT populations treated with nutlin-3 (5 µM) demonstrate that nutlin-3 induces a growth arrest in NIH cells, but not in NIHLT cells. Results presented are from one representative experiment (A) Cell cycle arrest was represented by the variation of ratio of arrested cells (G1+G2 phases) over proliferating cells (S phase) in treated cells versus untreated controls. (B) No variation of the percentage of cells in Sub-G1 phase, representing cell death, was observed after nutlin-3 treatment. (C–D) The use of a p53 inhibitor peptide (GSE22) validates the p53-dependence of the growth arrest induced by Nuclin-3. (C) High efficiency of infection and functionality of the GSE22 peptide were demonstrated by the accumulation of non-functional p53 in the nucleus by immunocytochemistry detecting p53 in NIH transduced with GSE22 or control vector. The stabilization of non-functional p53 was also seen in Western blots of the corresponding infected cells. (D) FACS analysis on NIH transduced with GSE22 or vector with nutlin-3 treatment (*<i>P</i><0.05, **<i>P</i><0.01 t-test).</p

Necdin confers resistance to p53-dependent growth arrest.

<p>(A–B) NIHLT cells depleted in Necdin by shRNAs and exposed to nutlin-3 showed an increase in growth arrest (A) measured by DNA content analysis by flow cytometry (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031916#pone-0031916-g004" target="_blank">figure 4A</a>) or (B) assessed by Wst-1 colorimetric assay. Results for Wst-1 represent normalized data according to the portion of arrested cells (O.D. untreated – O.D. treated) relative to arrested control NIH after 48 h of exposure to nutlin-3. (C–D) NIH and NIHLT cells overexpressing Necdin showed growth arrest resistance upon nutlin-3 treatment. (C) FACS analysis or (D) Wst-1 colorimetric assay (*<i>P</i><0.05, **<i>P</i><0.01 t-test).</p

Necdin expression is detected in LMP and is lower in TOV.

<p>Q-PCR analysis of tissues from seven LMP serous ovarian cancers and eight high grade serous ovarian cancers. Expression of Necdin gene (<i>P</i><0.0001, Mann-Whitney's U test) relative to ERK-1.</p

Functional analysis for a dataset of differentially expressed genes (≥1

5 fold) in OC spheroids following CT drugs treatments. A. Functional analysis following all drugs (cisplatin, topotecan and paclitaxel) treatment, B. Functional analysis following cisplatin treatment. Top functions that meet a -value cutoff of 0.05 are displayed.<p><b>Copyright information:</b></p><p>Taken from "Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids"</p><p>http://www.biomedcentral.com/1471-2164/9/99</p><p>BMC Genomics 2008;9():99-99.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2279123.</p><p></p

A) Phase contrast features of the monolayer-cultured EN-1078D cells revealing a sheet of polygonal cells with a pavement-like arrangement

<p><b>Copyright information:</b></p><p>Taken from "Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro"</p><p>http://www.rbej.com/content/5/1/38</p><p>Reproductive biology and endocrinology : RB&E 2007;5():38-38.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2092433.</p><p></p> B) Transmission electron microscopy showing the tendency of cells to pile up. C) Ultrastructural aspects of EN-1078D cell line by transmission electron microscopy. Cultured cells show a high nucleus-cytoplasmic ratio, euchromatic nuclei, prominent nucleoli and well-developed microvilli. D) Higher magnification shows intracytoplasmic lipid droplets (L), and exhibits intricate cytoplasmic interdigitations but no desmosomes were observed

Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids-2

Tering of OC spheroids following treatment with all used drugs (cisplatin, topotecan and paclitaxel (taxol)), that discriminates between compact spheroids and aggregates. A subset of candidate genes were initially obtained by filtering on signal intensity (2-fold), retaining 527 genes. One-way ANOVA parametric test (Welch -test, variances not assumed equal, ≤ 0.03) further selected 85 genes. Clustering analysis based on the 85 gene list was performed using the standard Condition Tree algorithm provided in GeneSpring. The mean appears , whereas signifies up-regulation, and signifies down-regulation (see legend bar). Compact spheroids are indicated in , aggregates are indicated in . Each cell line is indicated with different color.<p><b>Copyright information:</b></p><p>Taken from "Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids"</p><p>http://www.biomedcentral.com/1471-2164/9/99</p><p>BMC Genomics 2008;9():99-99.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2279123.</p><p></p

Network analysis of dynamic gene expression in OC spheroids based on the 1

5-fold common gene expression list obtained following treatment with all CT drugs used (cisplatin, topotecan and paclitaxel). The five top-scoring networks were merged and are displayed graphically as node (genes/gene product) and edges (the biological relationships between the nodes). Intensity of the node color indicates the degree of up- (red) or downregulation (green). Nodes are displayed using various shapes that represent the functional class of the gene product (square, cytokine, vertical oval, transmembrane receptor, rectangle, nuclear receptor, diamond, enzyme, rhomboid, transporter, hexagon, translation factor, horizontal oval, transcription factor, circle, other). Edges are displayed with various labels that describe the nature of relationship between the nodes: ---- binding only, → acts on. The length of an edge reflect the evidence supporting that node-to-node relationship, in that edges supported by article from literature are shorter. Dotted edges represent indirect interaction.<p><b>Copyright information:</b></p><p>Taken from "Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids"</p><p>http://www.biomedcentral.com/1471-2164/9/99</p><p>BMC Genomics 2008;9():99-99.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2279123.</p><p></p

Determination of EN-1078D sensitivity to cisplatin (left panel) and doxorubicin (right panel)

<p><b>Copyright information:</b></p><p>Taken from "Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro"</p><p>http://www.rbej.com/content/5/1/38</p><p>Reproductive biology and endocrinology : RB&E 2007;5():38-38.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2092433.</p><p></p> A, D) 24 hours of exposure to compounds B, E) 48 hours and C, F) 72 hours. Both Hela and EN-1078D are sensitive to cisplatin after 24 hours in contrast to KLE, which is chemoresistant. The three cell lines seem not to be affected by doxorubicin after exposure of 24 hours. KLE is more resistant than other cells lines for both 48 and 72 hours. Data shown are representative of results obtained from three independent assays