Fig. 3.

Effects of exogenous Wnt5a on Rho A activity and neuronal differentiation. NPCs were stimulated with Wnt5a (20 ng/ml) for 6 h, and mRNA levels of Nt3 and Ngn1 were analyzed by RT-PCR (a) and real time-RT-PCR (b). n = 3. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with the untreated control. c Cells were treated with Wnt5a (20 ng/ml) for 2 days, and then lysed and harvested. Western blotting was performed using anti-NT3, anti-Ngn1, or anti-calnexin to detect respective protein bands. Graphs show mean densities as fold change for three independent experiments (n = 3). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with untreated cells. d and e NPCs were treated with Wnt5a (20 ng/ml) for 3 days, and were stained with anti-Tuj1 to visualize neurite extensions. Scale bar, 20 μm. e Neurite lengths were measured in randomly selected fields using three independent experiments. n = 3 per group. Data are mean ± SD; Student’s t test. ** p < 0.01 compared with untreated control. f GTP-loaded RhoA activity was measured using a pull-down assay, as described in the Materials and Methods section, after treatment of cells for 15 min with Wnt5a (20 ng/ml). The data were normalized to the amount of total RhoA. Graphs show mean densities as fold change for four independent experiments (n = 4). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. *** p < 0.001 compared with the untreated control. g and h Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then treated for 6 h (g) or 2 days (h) with Wnt5a (20 ng/ml). g mRNA levels of Nt3 and Ngn1 were analyzed by real-time RT-PCR. n = 3. Data are mean ± SD; Student’s t test. * p < 0.05, ** p < 0.01 compared with control siRNA/Wnt5a. h Western blotting was performed using anti-NT3, anti-Ngn1, anti-RhoA, or anti-calnexin to detect the respective protein bands. i and j Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then treated for 3 days with Wnt5a (20 ng/ml). They were then stained with anti-Tuj1. Scale bar, 20 μm. j Neurite lengths were measured in randomly selected fields using five independent experiments. n = 5 per group. Data are mean ± SD; Student’s t test. *** p < 0.001 compared with control siRNA/Wnt5a