Figure 3

Effect of tenascin-C on proliferation (a) and adhesion (b and c) of hRPE cells and proliferation (d), adhesion (e and f), migration (g), and tube formation (h and i) of HMVECs. (a) hRPE cell proliferation was assessed by BrdU incorporation (mean±s.e.m.; n=4 per group). hRPE cell proliferation was significantly increased by tenascin-C in a dose-dependent manner. (b and c) hRPE cells were incubated with the indicated concentrations of tenascin-C or TGF-β2 (positive control) in fibronectin-coated 96-well plates for 1 h (mean±s.e.m.; n=4 per group) to analyze the cell adhesion. hRPE cell adhesion was significantly upregulated by TGF-β2 (10 ng/ml) and downregulated by tenascin-C (300 ng/ml). Photographs were taken with a 20 × objective after Hoechst 33342 staining. (d) HMVECs were incubated with the indicated concentrations of tenascin-C for 24 h to assess proliferation (mean±s.e.m.; n=4 per group). (e and f) HMVECs were incubated with the indicated concentrations of tenascin-C in fibronectin-coated 96-well plates for 12 h to assess adhesion (mean±s.e.m.; n=4 per group). (g) HMVECs stimulated with tenascin-C were placed in the upper chamber and allowed to migrate to the reverse side of the membrane to assess the cell migration (mean±s.e.m.; n=4 per group). (h and i) HMVECs were seeded on a basement membrane (BM) matrix with the indicated concentrations of tenascin-C (mean±s.e.m.; n=6 per group). Tubes formed by HMVECs were photographed after incubating for 12 h (4 × objective). (d–i) Tenascin-C significantly increased the HMVEC proliferation, adhesion, migration, and tube formation, in a dose-dependent manner. Values are expressed as a percentage relative to the untreated control. *P<0.05, **P<0.01, ANOVA/Dunnett’s test.