The WNK-OSR-GSK3ß pathway is involved in the neural development.

(A–B) siRNA-treated differentiated Neuro2A cells induced by RA for 24 h; (A) Control siRNA, (B) siGSK3ß. (C) The average length of neurites in siRNA-treated differentiated Neuro2A cells induced by RA for 24 h, shown in A and B (Control siRNA (n = 93), siGSK3ß (n = 91)). * p<0.0005 calculated by the Student’s t-test. (D) Gene expression determined by RT-PCR or quantitative RT-PCR analysis was examined in Neuro2A cells. Cells treated with siRNA against GSK3ß (siGSK3ß); (lanes 1 and 2) undifferentiated cells, (lanes 3 and 4) cells differentiated by RA for 24 h. The value obtained from each sample was normalized to that of GAPDH. The value of Lhx8, ChAT or Gad1 from differentiated cells under control siRNA treatment (lane 3) was set to 100. (E–H) Differentiated Neuro2A cells were transfected with various combinations of siRNAs and expression plasmids. (E) Control siRNA and control vector. (F) Control siRNA and GSK3ß. (G) siWnk1 and siWnk4, and control vector. (H) siWnk1 and siWnk4, and GSK3ß. (I) The average length of neurites in siRNA-treated differentiated Neuro2A cells induced by RA for 24 h, shown in E-H (Control siRNA and Control vector (n = 81), Control siRNA and GSK3ß (n = 87), siWnk1 and siWnk4, and Control vector (n = 103), siWnk1 and siWnk4, and GSK3ß (n = 77)). * p<0.0005 calculated by the Bonferroni correction. ns indicated non-significance. (J) Gene expression determined by RT-PCR or quantitative RT-PCR analysis was examined in Neuro2A cells. Cells were treated with various combinations of siRNAs and expression plasmids; (lanes 1–4) undifferentiated cells, (lanes 5–8) cells differentiated by RA for 24 h, (lanes 1–2 and 5–6) control siRNA, (lanes 3–4 and 7–8) siWnk1 and siWnk4, (lanes 1, 3, 5 and 7) control vector, (lanes 2, 4, 6 and 8) GSK3ß. The value obtained from each sample was normalized to that of GAPDH. The value of Lhx8, ChAT or Gad1 from differentiated cells under the treatment of control siRNA (lane 5) was set to 100.