Fig. 4

Effects of Wnt5a/RhoA/ROCK pathway on JNK activation and neuronal differentiation. a NPCs were treated with Wnt5a (20 ng/ml) for the indicated time duration, lysed and harvested. Western blotting was performed using anti-p-JNK, anti-JNK, or anti-calnexin to detect the 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 0 min. b and c Cells were pretreated with 2 μM SP600125 (SP) for 1 h and stimulated with Wnt5a (20 ng/ml) for 6 h. mRNA levels of NT3 and Ngn1 were analyzed by RT-PCR (b) and real-time RT-PCR (c). n = 3. Data are mean ± SD; Student’s t test. ** p < 0.01 as compared to Wnt5a-treated cells. d Cells were pretreated with 2 μM SP for 1 h and treated with Wnt5a (20 ng/ml) for 2 days. Western blotting was performed using an anti-NT3, anti-Ngn1, or anti-calnexin to detect the respective protein bands. Graphs show mean densities as fold change from three independent experiments (n = 3). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. * p < 0.05 compared with Wnt5a-treated cells. e and f Cells were pretreated with 2 μM SP for 1 h and treated with Wnt5a (20 ng/ml) for 3 days. They were then stained with anti-Tuj1. Scale bar, 20 μm. f Neurite lengths were measured in randomly selected fields using four independent experiments. n = 4 per group. Data are mean ± SD; Student’s t test. *** p < 0.001 compared with Wnt5a-treated cells. g Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and then treated for 30 min with Wnt5a (20 ng/ml). Western blotting was performed using anti-p-JNK, anti-JNK, anti-RhoA, or anti-calnexin to detect the respective protein bands. Graphs show mean densities as fold change from three independent experiments (n = 3). Band intensity was quantified with Quantity Ones® software. Data are mean ± SD; Student’s t test. * p < 0.05 as compared to control siRNA/Wnt5a. h Cells were pretreated with 5 μM Y27632 for 1 h and stimulated with Wnt5a (20 ng/ml) for 30 min. Western blotting was performed using anti-p-JNK, anti-JNK, or anti-calnexin to detect the respective protein bands. Graphs show mean densities as fold change from 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 Wnt5a-treated cells