22 research outputs found

    Outline of the gSFA procedure.

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    <p>The <i>gene Signature Finder Algorithm</i> consists of 4 separate steps: (1) find candidate seed genes; (2) generate a signature starting from each seed gene; (3) prune the signatures by statistical inference; (4) integrate signatures by gene ranking.</p

    Cross-talk between TNF-α and PPARγ signaling regulates DCBLD2 and NT5E intracellular levels in CRC cell lines.

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    <p><b>a</b>. Western blotting analysis shows PPARγ expression in HT29 and RKO CRC derived cell lines, as a model to investigate variations of NTE5 levels in response to TNF-α treatment. <b>b</b>. PPARγ-dependent protein induction of NT5E by TNF-α in CRC cells treated with 12,5 ng/ml TNF-α at different time points and analyzed by western blotting. <b>c</b> and <b>d</b>. Time course of <i>NT5E</i> and <i>DLBLC2</i> mRNA and protein regulation by troglitazone (TGZ), a PPARγ agonist, in the same cells. β-actin and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as control to normalize expression levels in Western blotting and Real-time RT-PCR analysis. The results are expressed as means ±S.D. of three independent experiments. <sup>*</sup><i>p</i> < 0.05<i>, <sup>**</sup>p</i> < 0.01.</p

    Analysis of the signatures generated by gSFA.

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    <p><b>a</b>. Dendrogram of the dichotomies generated by each of the 16 gSFA signatures (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072638#tab1" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072638#pone.0072638.s001" target="_blank">Figure S1</a>); <b>b</b>. Survival plot of the 133 samples stably classified in the good prognosis group, 56 samples in the poor prognosis group and 43 uncertain samples (log-rank test gives <i>p</i> < 10<sup>--16</sup>); <b>c</b>. heatmap of DEGs between the two stable groups, Red indicates overexpressed genes (expression levels over the median) and green indicates underexpressed genes (expression levels under the median); <b>d</b>. survival curves samples in the two clusters of the heatmap as in <b>c</b>. (<i>p</i> = 6.6 ·10<sup>-6</sup>).</p

    TMAs and western blot validation analysis of AKAP12, DCBLD2, NT5E and SPON1.

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    <p><b>a</b>. “Columns from left to right” indicate immunostaining pattern in normal colonic samples and CRC cores negative and positive for each marker AKAP12, DCBLD2, NT5E, SPON1. Black arrows indicate immunohistochemical staining pattern in normal or malignant colonic cells. White arrows indicate the immunostaining distribution in the stromal compartment (endothelial, regulatory T-cells and macrophages) characteristic of NT5E expression pattern. Magnification 10X; <b>b</b>. Number of positive cases detected on TMA validation series comprising tumor specimens (TUM) and a subgroup of matched normal colonic mucosa (NM). Error bars indicate the standard deviation from the mean (<i>p</i> < 0.05); <b>c</b>. Four representative frozen CRC specimens (T) and matched normal mucosa (N) were identified in the same cohort of patients and analyzed by immunoblot. Molecular weight markers are indicated in kilodaltons. β-tubulin was used as loading control to normalize band intensities.</p

    Array analysis of genes regulated by the PI3K/AKT pathway in lung epithelial cells.

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    <p>(A) Heatmap representation of expression values of DEGs comparing BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN to BEAS-C control cells. Hierarchical clustering analysis was perfomed with Euclidean metric distance and Ward’s linkage rule. (B) The Venn diagram shows the number of common, specific and exclusive DEGs.</p

    Analysis of DEG expression in BEAS-C and derivatives cells treated with pharmacological inhibitors of the PI3K/AKT pathway.

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    <p>BEAS-C cells and derivatives were treated for 24 hours with the indicated doses of MK2206 and LY294002, respectively. <b>(A)</b> The graphs show the mRNA levels (mean ± SD) of the indicated up-regulated genes in BEAS-2B cells and derivatives. <b>(B)</b> The graphs show the mRNA levels (mean ± SD) of the indicated down-regulated genes in BEAS-2B cells and derivatives. Bars are mean ± SD of 3 independent experiments conducted in triplicate, statistical analysis that was performed by Two-way ANOVA and Dunnett's test, p-values as indicated <b>(C)</b> Immunoblot analysis of Phospho-AKT (Ser 473), AKT1 and actin in BEAS-C cells and derivatives.</p

    Correlation between the activation status of the PI3K/AKT pathway and DEG expression in NSCLC cell lines.

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    <p><b>(A)</b> Immunoblot analysis of phosphorylated AKT in NSCLC cell lines. AKT1 and actin were used to normalize AKT phosphorylation and protein loading, respectively. <b>(B)</b> The graph shows mRNA levels of up-regulated DEGs (GDF15, PTGES, S100P) in NSCLC cell lines. Statistical analysis was performed on 3 independent experiments conducted in triplicate and was performed by One-way ANOVA test for each gene relative to each cell line, p-values as indicated <b>(C)</b> The graph shows mRNA levels of three representative down-regulated DEGs (SGK1, IGFBP3, PEG10) in NSCLC cell lines. Bars are mean ± SD of a representative experiment. Statistical analysis was performed on 3 independent experiments conducted in triplicate and was performed by One-way ANOVA test for each cell line, p-values as indicated.</p
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