Morphological and functional modifications induced by CD157 knockdown in OV-90 cells.

<p>(A) sqRT-PCR (left) and western blot analysis (right) showing OV-90 cells retrovirally transduced with a shRNA that targets the human CD157 mRNA, resulting in efficient knockdown of CD157 expression. GAPDH and β-actin were used as internal controls, respectively. (B) Morphology of colonies formed by OV-90/scramble and OV-90/shCD157 cells. Representative colonies visualized after crystal violet staining are shown. Scale bar: 200 µM. (C) sqRT-PCR for E-cadherin, N-cadherin and Snail, Twist1 and Slug transcriptional repressors in OV-90/scramble and OV-90/shCD157 cells. Densitometry quantifies the levels of mRNA expression of the indicated molecules relative to GAPDH. (D) Anoikis assay. After 48, 72, 96 and 192 h under anchorage-independent growth, cells were fixed, stained with propidium iodide and analyzed with a FACSCanto. Data analysis was performed with ModFit LT™ cell cycle analysis software. Anoikis in OV-90/scramble and OV-90/shCD157 cells was determined by measuring the percent of sub-G1 cells. Results represent the mean ± SEM of three independent experiments. *P<0.05; **P<0.01, two-tailed t test. (E) Anchorage-independent growth of OV-90/scramble and OV-90/shCD157 cells was analyzed by soft agar colony formation assay. Graph represents the average number of colonies/field formed from three independent experiments ± SEM after 3 weeks incubation of cells in soft agar. *P<0.05, two-tailed t test. (F) Effect of CD157 knockdown on OV-90 cell migration in a scratch-wound assay. Cells were grown as monolayers, wounded, and photographed at time 0 and at 24 h (scale bar: 200 µM). Wound edges are indicated by black dashed lines. (G) The ability of OV-90/scramble and OV-90/shCD157 cells to close the wound was calculated by measuring 20 randomly chosen distances along the wound edge at time 0 and at 24 h. Results represent the percentage reduction of the average wound width and are expressed as the mean ± SEM of three independent experiments. **P<0.01, two-tailed t test.</p

CD157 overexpression protects OV-90 cells from anoikis and enhances motility.

<p>(A) sqRT-PCR (left) and western blot analysis (right) of CD157 in OV-90/CD157 and OV-90/mock cells. GAPDH and β-actin were used as internal controls, respectively. (B) Morphology of colonies formed by OV-90/mock and OV-90/CD157 cells. Representative colonies visualized after crystal violet staining are shown. Scale bar: 200 µM. (C) sqRT-PCR analysis of E-cadherin and N-cadherin in OV-90/mock and OV-90/CD157 cells. Densitometry quantifies the levels of mRNA expression of the indicated molecules relative to GAPDH. (D) Effect of CD157 overexpression on anoikis. After 48, 72. 96 and 192 h of anchorage-independent growth, cells were fixed, stained with propidium iodide and analyzed with a FACSCanto. Anoikis in OV-90/mock and OV-90/CD157 cells was determined by measuring the percent of sub-G1 cells. Results represent the mean ± SEM of three independent experiments. *P<0.05; **P<0.01, two-tailed t test. (E) Anchorage-independent growth of OV-90/CD157 and mock cells was analyzed by soft agar colony formation assay. Graph represents average number of colonies formed from three independent experiments ± SEM after 3 weeks incubation of cells in soft agar. ***P<0.001, two-tailed t test. (F) Effect of CD157 expression on cell migration in a scratch-wound assay in OV-90/CD157 and mock cells. Cells were grown as monolayers, wounded, and photographed at time 0 and at 24 hr. Wound edges are indicated by black dashed lines (scale bar: 200 µM). (G) The ability of cells to close the wound was calculated by measuring 20 randomly chosen distances along the wound edge at time 0 and at 24 hr. Results represent the percentage reduction of the average wound width and are expressed as the mean ± SEM of three independent experiments. *P<0.05; two-tailed t test.</p

Gene ontology analysis of genes modulated by CD157 overexpression.

<p>Go analysis of differentially induced or repressed genes shared by OVCAR-3/CD157 and OV-90/CD157 cells with respect to enrichment of genes with assignments to specific biological processes. The number of genes in a specific biological process is indicated in brackets.</p

High CD157 expression influences spheroid formation and disaggregation in OV-90 cells.

<p>(A) Representative morphology of spheroids formed by OV-90/mock (top) and OV-90/CD157 (bottom) (scale bar: 100 µM). (B) Phase contrast microscopy images of spheroid disaggregation on fibronectin. A representative spheroid of OV-90/mock and OV-90/CD157 cells at time 0 and at 12 h are shown (scale bar: 200 µM). (C) Spheroids were photographed at time 0 and at 12 h, and the increase in the size of disaggregation area calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043649#s2" target="_blank">Materials and Methods</a> is reported. The means ± SD of the fold change in area of 20 spheroids per condition of a representative experiment, repeated (n = 3), are shown. ***P<0.001, two-tailed t test.</p

CD157 overexpression alters the expression of epithelial and mesenchymal markers.

<p>(A) Confocal microscopy analysis of E-cadherin and (B) β-catenin expression in OVCAR-3/CD157 and mock cells. Cells were grown on a gelatin-coated coverslip, fixed, permeabilized and stained with anti-E-cadherin, and anti-β-catenin antibodies followed by secondary Alexa Fluor-488-labelled antibody. Samples were analyzed with an Olympus FV300 laser scanning confocal microscope and by Nomarski differential interference contrast (DIC) optics. For E-cadherin, a fluorescence image merged with DIC image is shown (scale bar: 50 µM). Semiquantitative analysis of E-cadherin junctional staining was determined by counting a minimum of 10 fields/sample (at least 200 cells overall) and scoring as positive cells with two remaining fluorescent intercellular borders. For β-catenin, a fluorescent image is shown. Inset: amplified view of an individual OVCAR-3/CD157 cell exhibiting diffuse β-catenin staining in the plan of focus cutting through the nucleus. Asterisks correspond to nucleoli. (C) Western blotting for E-cadherin and β-catenin in OVCAR-3/CD157 and mock cells. Densitometry quantifies the expression level of E-cadherin and β-catenin relative to β-actin. (D) β-catenin levels in nuclear and cytoplasmic fractions of OVCAR-3/mock and OVCAR-3/CD157 cells were determined by western blot analysis. α-tubulin and lamin B1 (LamB1) were used as cytoplasmic and nuclear loading controls, respectively. Densitometry quantifies the expression level of β-catenin relative to the proper control. (E) sqRT-PCR for E-cadherin, N-cadherin, β-catenin and for E-cadherin transcriptional repressors in OVCAR-3/CD157 and mock cells. Densitometry quantifies the levels of expression of E-cadherin repressors relative to GADPH. (F) qRT-PCR for Zeb1, Snail and Twist1. The comparative CT method was used to determine gene expression in CD157-transfected cells relative to the value observed in the mock cells, using TBP as normalization control. Histograms report the means ± SEM of three qRT–PCR independent experiments, each conducted in triplicate. *P<0.05, ***P<0.001; <i>ns</i>, not significant; two-tailed t test. (G) Western blot analysis showing the level for Snail and Zeb1 in OVCAR-3/mock and OVCAR-3/CD157 cells. Densitometry quantifies the expression level of both proteins relative to β-actin. Results shown in each panel are representative of three independent experiments with similar results.</p

Gene expression profiling of OVCAR-3 and OV-90 cells overexpressing CD157.

<p>(A) Hierarchical clustering applied to the expression matrix of genes similarly regulated in both OVCAR-3 and OV-90 cells overexpressing CD157, using Euclidean distance as similarity metrics and complete linkage as the linkage method. A red-to-green gradient was used to indicate, for each gene, levels of up- or down-regulation. (B) Dot plot shows 378 significantly modulated genes (163 up-regulated and 215 down-regulated) shared by OVCAR-3/CD157 and OV-90/CD157 cells. Single genes are indicated by red (up) and green (down) data points. (C, D) A panel of modulated genes was selected and validated by qRT-PCR. (C) Fold changes of the various indicated genes in OVCAR-3 and OV-90 cells following CD157 overexpression are shown. (D) qRT-PCR validation of the genes shown in panel C. The comparative CT method was used to determine gene expression in CD157-transfected cells relative to the value observed in the mock-transfected cells, using TBP as normalization control. Histograms report the means ± SD of a qRT–PCR experiment conducted in triplicate. (E) The expression of VCAN, EpCAM and BMP7 was examined in OVCAR-3/CD157, OV-90/CD157 and the corresponding control cells by western blot analysis. Densitometry quantifies the expression level of the indicated proteins relative to β-actin. Results shown are representative of three independent experiments with similar results.</p

Additional file 8: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Table S5. Univariate analysis results, full model multivariate analysis results, as well as post hoc power calculations. (DOCX 14 kb

Additional file 2: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Table S1. Primers used in the study. (DOCX 12 kb

Additional file 1: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Figure S1. Experimental design. (DOCX 18 kb

Additional file 5: of X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Figure S2. Sodium bisulfite sequencing methylation analysis of 51 CGs across the ZPBP2 promoter region shows higher methylation levels in fibroblast cell lines with two and three copies of the Xq arm. A: location of the 51 CGs interrogated using the sodium bisulfite sequencing assay shown in the context of the UCSC browser (hg19). B: heatmap representing mean methylation levels for each of the 51 CGs in cell lines with one X chromosome (two fibroblast cell lines with karyotype 45,X and no SRY region) and two or three copies of Xq (data from four fibroblast cell lines with karyotype 46,XX and two fibroblast cell lines from Turner syndrome patients with karyotype 46,i(Xq)). The black box beneath the heatmap shows the location of the 10 CGs analyzed by the pyrosequencing assay. The color scale for percent methylation is shown on the right. (DOCX 292 kb