Figure 4

Flow cytometric analysis of syndecan-2 surface expression on HCT-116 cells. (A) HCT-116 cells were cultured for 48 h in the absence of matrix (Ctrl), in the presence of stromal fibroblast ECM (Fibrob. ECM) or in the presence of their own matrix (HCT-116 self ECM) and then immunostained with anti-syndecan-2 antibody. (B) The relative staining was determined by densitometric analysis. (C) Fibroblasts were immunostained with anti-fibronectin antibody, anti-laminin antibody or anti-collagen antibody to confirm the presence of these proteins. The photo shows images that were obtained using a confocal microscope. HCT-116 cells were cultured for 72 h on fibronectin, laminin or collagen I and labeled with anti-syndecan-2 antibody. (D) Fibroblasts were cultured in Petri dishes until confluence and its ECM was extracted as described in Methods. Total protein from the ECM (Fibro. ECM) was then applied to polyacrylamide gel 7.5% with 10μg of standard fibronectin (P). After transfer, the nitrocellulose membrane was incubated with anti-fibronectin and revealed with DAB. Collagen and laminin were not detected on ECM produced by fibroblasts through Western blotting. (E) HCT-116 cells were cultured in the presence of collagen-I, laminin or fibronectin for 72 h and then stained with anti-syndecan-2 antibody and the relative levels of this protein were determined by densitometric analysis (F). (G) HCT-116 cells were cultured in the absence or presence of fibronectin for different lengths of time (12, 24, 48 and 72 h) and then stained with anti-syndecan-2 antibody and the relative staining was determined by densitometric analysis (H). The gray peak represents the control cells cultured in the absence of ECM, the black line represents cells cultured in the presence of ECM and the gray line represents the control for the secondary antibody, anti-IgG. *p ≤ 0.05 compared to control.