Effects of SRPK1-knockdown on cell cycle progression in SKOV3 cells.

<p>(A) Cell cycle progression from G2/M-arrest. Cells at 80% confluence were arrested in G2/M phases with nocodazole (NOC, 600 ng/ml) for 12 hr, washed with phosphate buffered saline (PBS), and released into medium without drug. Cells were collected at the indicated times and subjected to propidium iodine staining and flow cytometric analysis. Representative data obtained from two independent experiments are shown. (B) Cell cycle progression from G0/G1-arrest. Cells at 100% confluence were incubated in medium without serum. Quiescent cells were activated with 10% FBS-containing medium. Cells were collected every 3 hr for flow cytometric analysis as described above. The percentage of cells in G1, S, and G2/M was determined using Modfit program and a histogram graph was generated using the WinList program (Verity Software House). Representative data obtained from two independent experiments are shown.</p

Forced inhibition of SRPK1 expression in ovarian cancer cell lines enhances sensitivity to cisplatin.

<p>Ovarian cancer cells were transiently (A) or stably (C) transfected with either the siRNA-encoding shSRPK1 plasmid or the empty vector (pSM2-EV). Protein levels of SRPK1, UPF1 and actin were determined by Western blot analysis. Representative blots from three independent experiments are shown. (B) and (D) SRPK1 knockdown enhances cisplatin cytotoxicity. (B) Cells (5×10⁴) were reseeded 24 h after transfection, treated with various concentrations of cisplatin for 48 hr and the number of surviving cells was analyzed by MTT assay. Survival (%) is expressed relative to non-treated pSM2-EV cells. (D) Stable transfectants (3×10²) were seeded in triplicate, treated with cisplatin for 24 hr and colony formation was assessed after 10–14 days. Data were analyzed with one way ANOVA and * indicates P<0.05; bars, SD.</p

Phosphorylation patterns of SR-proteins and MAPK42/44 and AKT proteins are altered in SRPK1-knockdown SKOV3 cells.

<p>Western blot analysis was performed on lysates prepared from SKOV3-derived pSM2-EV cells and two stable SRPK1-knockdown clones. Antibodies were used to detect the phosphorylated SR proteins (mAB104), MAPK42/44 (Thr²⁰²/Tyr²⁰⁴), and AKT (Ser^473/Thr³⁰⁸) as well as total protein levels of MAPK42/44, and AKT.</p

SRPK-knockdown ovarian cancer cells exhibit reduced cell proliferation, anchorage-independent growth and <i>in vitro</i> migration ability.

<p>(A) Cell proliferation assay. Cells (1×10⁴) were seeded in triplicate in a 24-well plate, trypsinized and counted in the presence of trypan blue at the indicated times. (B) Anchorage-independent growth assay. Cells (5×10³) were seeded in soft agar and colony number was determined 21 days later. Representative fields of the colonies in soft-agar plates are also shown. Data shown are the mean of three independent experiments and were analyzed with paired t-test; * indicates P<0.05, bars, SD. SKOV3 cells were seeded as a positive control. (C) <i>In vitro</i> wound healing assay. Confluent cells were scratched with a 200 µl-tip to generate straight line-gaps. Images of the gaps were taken at 0 and 28 hr and the width of the gaps (white dashed-lines) was measured under a light microscope to calculate the rate of cell migration. Representative images are shown on the left and relative migrating distances (wound closure) shown on the right were calculated from 20 areas from each plate. Data shown are from three independent experiments. <i>Asterisks</i> show significant differences compared with control (<i>P</i><0.05).</p

DNA hypomethylation-mediated activation of <i>Cancer/Testis Antigen 45</i> (<i>CT45</i>) genes is associated with disease progression and reduced survival in epithelial ovarian cancer

<div><p>Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. <i>CT45</i> was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased <i>CT45</i> expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with <i>CT45</i> expression in the TCGA database for EOC. <i>CT45</i> promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. <i>CT45</i> expression was increased and <i>CT45</i> promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. <i>CT45</i> hypomethylation was directly associated with <i>LINE-1</i> hypomethylation, and <i>CT45</i> was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses <i>CT45</i> promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.</p></div

Enriched GO Biological Processes for the genes with significant associations with GWAS discovered variants.

<p>Enriched GO Biological Processes for the genes with significant associations with GWAS discovered variants.</p

Significant associations between <i>rs2072590</i> genotype and <i>MLH1</i> expression phenotypes (permutated P = 0.0049, adjusted r² = 8.3%).

<p>The boxplot shows the relationship between log₂ residuals of <i>MLH1</i> expression levels (adjusted for age and case-control status) and genotype of the <i>rs2072590</i>.</p

List of Top ranked significant associations between mRNA and variants (P<0.001).

*<p>Permutated P value.</p