001) and collagen I (ANOVA p = 0.04). Results are expressed as absorbance at 405 nm with a reference wavelength of 620 nm. Data shown is mean ± standard deviation (n = 3). Student’s t -test; p ≤ 0.05*, 0.01**, 0.005***. The more invasive Clone #3, displays significantly decreased adhesion to matrigel (p = 0.01), laminin (p = 0.02), fibronectin (p = 0.01) and collagen type IV (p = 0.01) compared to the parental cell line (Fig 2B). In contrast a significant increase in adhesion was observed to collagen type I (p = 0.003), although the level of adhesion to the collagens was significantly HKI-272 datasheet lower than that to fibronectin or laminin. The less invasive Clone

#8, showed significantly increased adhesion to matrigel (p = 0.04) and laminin (p = 0.002). Adhesion to fibronectin and collagen type I were also increased, but not significantly and adhesion to collagen type IV was decreased significantly (p = 0.001) for Clone #8. Anoikis and anchorage-independent growth The evaluation of survival in suspension (anoikis) showed that Clone #3 was resistant to anoikis compared to the parental cell line, although this difference did not reach statistical significance (p = 0.07). Clone #8 demonstrated a significant sensitivity to anoikis (p = 0.02) compared

to the parental cell line, MiaPaCa-2 (Fig 3A). Anchorage-independent growth was assessed using the soft agar assay. MiaPaCa-2 showed colony formation with an average colony

size of 75 μm and percentage colony forming https://www.selleckchem.com/products/pci-34051.html efficiency (% CFE) of 48%; Clone #3 formed more and larger colonies with an PI3K/Akt/mTOR inhibitor average Staurosporine price size of 120 μm and a %CFE of 69%. In contrast, Clone #8 (low invasion and high adhesion), showed significantly reduced ability (32% CFE) to form colonies (p = 0.006) and the average size of colonies was 60 μm (Fig 3B). Figure 3 A. Percentage survival of MiaPaCa-2, Clones #3 and Clone #8 in suspension compared to adherent cells, ANOVA ( p = 0.002). B. Percentage colony formation efficiency (%CFE) of MiaPaCa-2, Clone #3 and Clone #8 under anchorage-independent growth conditions, ANOVA (p = 0.02). Data shown is mean ± standard deviation (n = 3). Student’s t -test; p ≤ 0.05*, 0.01**, 0.005***. Integrin expression Significant changes in invasion and adhesion to fibronectin and laminin were observed in the sub-populations. Therefore, expression of integrins β1, α5 and α6, which are associated with adhesion to laminin and fibronectin were examined in the cell lines, by immunoblotting (Fig 4A-C). Beta-actin used as loading control (Fig 4D). Compared to MiaPaCa-2, Clone #8 showed higher expression of integrins β1 and α5. Low levels of α6 were detected in Clone #8, while it was undetectable in the parental MiaPaCa-2 cells. Lower levels of each of the integrins were detected in Clone #3 compared to Clone #8. Figure 4 Immunoblot of A. Integrin β1 B. Integrin α5 C. Integrin α6 and D.