Additional file 3: Figure S2. of Non contiguous-finished genome sequence and description of Microbacterium gorillae sp. nov.

Gel view comparing Microbacterium gorillae strain G3T spectra with other members of the genus Microbacterium. The gel view displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a gray-scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. Displayed species are indicated on the right. (PPTX 76 kb

Additional file 4:Â Folder S1. of Non contiguous-finished genome sequence and description of Microbacterium gorillae sp. nov.

Annotation results. (RAR 1566 kb

Additional file 1: Table S1. of Non contiguous-finished genome sequence and description of Microbacterium gorillae sp. nov.

Differential phenotypic characteristics between Microbacterium gorillae sp. nov. strain G3T and others Microbacterium strains. (DOCX 14 kb

Inhibition of RUNX2 Transcriptional Activity Blocks the Proliferation, Migration and Invasion of Epithelial Ovarian Carcinoma Cells

<div><p>Previously, we have identified the RUNX2 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from serous epithelial ovarian cancer (EOC) patients, when compared to primary cultures derived from matched primary (prior to CT) tumors. However, we found no differences in the RUNX2 methylation in primary EOC tumors and EOC omental metastases, suggesting that DNA methylation-based epigenetic mechanisms have no impact on RUNX2 expression in advanced (metastatic) stage of the disease. Moreover, RUNX2 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. Knockdown of the RUNX2 expression in EOC cells led to a sharp decrease of cell proliferation and significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as various genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX2 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced.</p><p>Taken together, our data are indicative for a strong oncogenic potential of the RUNX2 gene in serous EOC progression and suggest that RUNX2 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX2 and other members of the RUNX gene family in ovarian tumorigenesis.</p></div

BSP analysis of the methylation status of RUNX2 in grade 3 primary serous EOC tumors compared to omental metastases.

<p>Filled circles represent methylated CpGs and open circles represent unmethylated CpGs. CpG plot of the analyzed region is also presented (CpGs are displayed with vertical marks). The indicated positions on the CpG plot represent the number of nucleotides stretching upstream of the first exon of the RUNX2 gene.</p

Selected differentially expressed gene groups in SKOV3 cells upon RUNX2 knockdown.

<p>Selected differentially expressed gene groups in SKOV3 cells upon RUNX2 knockdown.</p

ShRNA-mediated knockdown of the RUNX2 expression in SKOV3 cells.

<p>A, effect on cell proliferation; B, Representative images of colony forming assays following RUNX2 knockdown. Error bars denote ± SEM; *indicates statistical significance (<i>P</i><0.05).</p

Analysis of RUNX2 expression in SKOV3 cells.

<p>A. Semi-quantitative duplex RT-PCR analysis of RUNX2 mRNA expression levels in the shRNA-RUNX2 clones 3 and 6, compared to the mock-transfected control clone. Displayed are images of representative results following sqRT-PCR analysis. The 18S ribosomal RNA gene was used as internal standard. B. Western-blot analysis of RUNX2 protein expression in the shRNA-RUNX2 clones cl-sh3 and cl-sh6, compared to the mock-transfected clone (ctrl). β-actin was used as a loading control. C. Densitometric analysis of RUNX2 mRNA/protein expression levels in the clones sh3 and sh6, compared to the control. Differences between the control clone and shRNA-RUNX2 clones were determined by a Student's t-test. Error bars denote ± SEM; *indicates statistical significance (<i>P</i><0.05).</p

Effect of RUNX2 knockdown on SKOV3 cell migration and invasion.

<p>A. Migration was assessed using Boyden-chamber assay. Cells from the shRNA-RUNX clones 3 and 6 and the control clone were seeded into the upper chambers in 0.1% FBS containing medium at a density of 2.5×10⁴ per well, and 600 µl of 1% FBS containing medium was placed in the lower chamber as a chemoattractant. After 24 h at 37°C in 5% CO₂, the cells were fixed with cold methanol and stained with blue trypan solution. Migrated cells on the underside of the filter were photographed and counted by phase contrast microscopy. B. Cell invasion was assayed in a similar way, as the upper chambers were coated with Matrigel. Here, NIH3T3 conditioned medium was added in the lower chamber as a chemoattractant (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074384#s2" target="_blank">Materials and Methods</a> for details). All experiments were performed in triplicate. For each experiment, cell number was calculated as the total count from 10 random fields per filter (at magnification ×40). Differences between shRNA-RUNX2-transfected and vehicle-transfected SKOV3 cells were determined by a Student's t-test, where <i>p</i><0.05 was considered significant.</p

Functional analysis for a dataset of differentially expressed genes (≥2-fold) following RUNX2 suppression in SKOV3 cells.

<p>A. Functional analysis of upregulated genes; B. Functional analysis of downregulated genes. Top functions that meet a <i>p</i>-value cutoff of 0.05 are displayed.</p