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

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

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

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