Figure 4.

Erk signaling acts as negative regulator for the nanotube culture-induced chondrocytic transition. (A) Wing limb mesenchymal cells were cultured on Ti-control (flat titanium) and TiO₂ nanotube surfaces having different inner diameters. Changes in the protein levels of Erk and pErk were examined by Western blotting. HSP70 and GAPDH were detected as a loading control. (B) Wing limb mesenchymal cells were incubated with/without 500 nM AKT inhibitor IV, 10 µM PD98059, or 10 µM SB203580 and the expression and phosphorylation levels of p38 MAPK, Erk, and AKT was examined by Western blotting (upper panel). Wing limb mesenchymal cells were cultured on Ti-control (flat titanium) and 30-nm-diameter TiO₂ nanotube surfaces in the presence or absence of 500 nM AKT inhibitor IV, 10 µM PD98059, or 10 µM SB203580, and immunolabeled with Alexa-488-conjugated phalloidin and an anti-vinculin antibody (lower panel). (C) Changes in the protein levels of type II collagen were examined by Western blotting. HSP70 was detected as a loading control. The data shown are representative of at least four independent experiments.