Fibrosarcoma cell adhesion and spreading.

<p><b>a</b>): Cell adhesion to uncoated plastic or plastic coated with collagen I, fibronectin or laminin-1 at 45 min. * and ** represent significant differences, (<b>*</b>p<0.05 and **p<0.01) by two way ANOVA followed by Bonferroni post-test. <b>b</b>): Cell spreading on fibronectin and laminin at 30 min, on a thin layer of collagen at 3 h or on collagen for 3 h in the presence of 2% FCS (collagen+S). Cells were stained live with with CellMask orange. Scale bars, 50 µm. <b>c</b>): Immunoblot analysis for β1 integrin (β-Itg) ILK and N-cadherin. Blots were normalised to actin. Results are representative of at least 3 independent experiments.</p

Migration of fibrosarcoma cells expressing single VEGF isoforms.

<p><b>a</b>): Wound closure was measured at intervals up to 24 h. Each point represents data obtained from 2–5 independent experiments ± SEM; <b>b</b>): representative culture images of the four fibrosarcoma cell lines showing wound closure at 18 h.</p

Analysis of morphology and migration of fibrosarcoma cells exposed to contractility inhibitors.

<p><b>a</b>): Cells were treated with P6 (5 µM), blebbistatin (5 µM) or Y27632 (10 µM) for 18 h. Representative images of P6 treated cells by phase contrast microscopy; blebbistatin-treated cells stained for F-actin; Y27632 treated cells stained with CellMask Orange. Scale bars, 50 µm. <b>b</b>): Migration was quantified by measuring wound closure. Images were collected at 12 h for the fswt and fs188 cells, at 18 h for the fs120 cells and at 24 h for the fs164 cells to correlate with 40–60% wound closure in untreated conditions. Contractility inhibitors were added at the same concentrations used in (<b>a</b>) 18 h before the start of the assay and replaced in fresh media at assay start. Each point represents data obtained from 3 independent experiments ± SEM, each conducted with 2–4 replicates. *p<0.05, ***p<0.001 values represent significance over untreated cultures (ANOVA followed by Tukey-Kramer post-test). <b>c</b>): Cells were incubated overnight with P6 (5 µM). Equal amounts of proteins (30 µg) were analysed for integrin β1 (β-Itg) and pMLC. A representative immunoblot is shown.</p

Schematic of proposed signalling interactions and morphological and motility characteristics of fibrosarcomas expressing single VEGF isoforms.

<p>Differences in PI3K/AKT, Ras/Raf/MEK/ERK and JAK/Stat3 signalling between fswt/fs188 and fs164/fs120 cells are shown. Upregulation/downregulation of activities or levels of expression of individual proteins are indicated by up or down arrows. VEGF188 expression is associated with reduced proliferation rates, increased apoptosis and typical integrin-dependent mesenchymal morphology and migration mode. VEGF164/VEGF120 expression is associated with faster proliferation rates, increased survival and rounded/amoeboid integrin-independent morphology and mode of motility. Migration of fs164/fs120 but not fswt/fs188 cells is contractility-dependent.</p

Signalling pathway analysis in fibrosarcomas expressing single VEGF isoforms.

<p><b>a–d</b>): Where indicated cells were incubated overnight with JAK inhibitor P6 (5 µM), or PI3K inhibitor LY20990 (10 µM). Equal amounts of proteins (10–30 µg) were analysed for <b>a</b>): phospho-AKT (p-AKT) and p70S6; <b>b</b>): pGSK3, pFOXO1, p27 and p21; <b>c</b>): phospho-ERKs1/2 (p-ERK) and total ERK (t-ERK); <b>d</b>): phospho-Stat3 (p-Stat3). <b>e</b>): Proteins extracted from solid tumours (50 µg/lane) and analysed for pStat3. All blots were normalized with actin or GAPDH and are representative of at least three independent experiments. <b>f</b>): Concentrated conditioned media normalised against cell numbers, analysed for LIF expression.</p

Population doubling times (hours) of fibrosarcoma cells grown on 2D surfaces.

<p>Population doubling times (hours) of cells grown on plastic, collagen or fibronectin are means of 3–6 independent experiments ± SEM.</p><p><b>*</b>p<0.05,</p><p>**p<0.01,</p><p>***p<0.001 values represent differences between fswt/fs188 cells versus fs164/fs120 cells (two way ANOVA followed by Bonferroni post-test).</p