GFAP initially appeared at 72 h for cells grown on 50-nm nanodots (Figures 6 and 7a). Decrease of GFAP expression was observed
in cells grown on 100- and 200-nm nanodots for 72 h (Figure 7a). The effects of topography on the astrocytic processes were also observed. The 10-, 50-, and NU7026 ic50 100-nm nanodots induced longer astrocytic processes after 120 h of incubation (Figure 7b). Figure 6 Immunostaining of vinculin (green) and GFAP (red) in C6 glioma cells. The cells are seeded on nanodot arrays and incubated for 24, 72, and 120 h. Images are obtained using a confocal microscope. The scale bars indicate 25 μm. Figure 7 The GFAP-stained area, total length of glial processes, and the vinculin-stained area. (a) The GFAP-stained area per cell is plotted against the nanodot diameters and grouped by incubation time. (b) Total length of glial processes per PF-4708671 cell is plotted against the nanodot diameters and grouped by incubation time. Maximum process length occurs when cells are grown on 50-nm nanodots with 120 h of incubation. (c) Z-VAD-FMK chemical structure The vinculin-stained area per cell is plotted against the nanodot diameters and grouped by incubation time. Maximum staining occurs for cells grown on 10- and 50-nm nanodots. All values are expressed as the mean ± SD averaged from
at least six experiments. **p < 0.01, *p < 0.01. Vinculin is a membrane cytoskeletal protein associated with focal adhesion plaques that is involved in the linkage of integrin adhesion molecules Verteporfin chemical structure to actin filaments . The area of focal vinculin plaques significantly increased in the 10- and 50-nm nanodot-treated
groups at 24, 72, and 120 h (Figure 7c). Nanotopography enhanced connexin43 transport Nanodot arrays control astrocyte-astrocyte interaction by regulating the function of gap junction proteins. Cx43, which composes gap junction channels (GJCs), mediates transmission and dispersion growth/suppressive factors and reveals the contact spots between astrocytes [19, 20]. The expression level of Cx43 did not show a consistent pattern regarding the dot diameter (Figure 8). The 10-nm nanodots decreased the expression of Cx43 at 24 h. The Cx43 expression level significantly increased for cells grown on 50-nm nanodots for 72 h. Figure 8 Quantitation of connexin43 expression in C6 glioma cells grown on nanodot arrays. (a) Western blotting of C6 glioma cells with anti-Cx43 antibody. GAPDH staining serves as a control. (b) Expression of Cx43 relative to GAPDH is plotted against the nanodot diameters and grouped by incubation time. Values are expressed as the mean ± SD averaged from at least three independent experiments. *p < 0.05. Nanotopography modulated the expression and transport of Cx43 protein Immunostaining was used to obtain the expression and cellular localization of Cx43 in C6 glioma cells on nanodot arrays.