Fig. 4.

Vangl2 alterations affect cell growth, differentiation and active β-catenin expression in neural stem cells in vitro. a siRNA against Vangl2 induced a significant decrease of cell viability compared to control cells transfected with a control siRNA sequence in C17.2 cells (one-way ANOVA with Bonferroni post-test, control vs siRNA Vangl2 P = 0.0004). siRNA against Prickle1, siRNA against β-catenin, cDNA for Prickle1 or cDNA for Vangl2 caused no change in cell viability. b The mRNA expression of Prickle1 after siRNA or cDNA transfection of Prickle1, Vangl2 or β-catenin. Only cDNA Prickle1 and siRNA β-catenin induced significant changes in Prickle1 mRNA expression (one-way ANOVA with Bonferroni post-test, control vs cDNA Prickle1 P < 0.0001, control vs siRNA β-catenin P = 0.0043). The mRNA expression of Vangl2 was below the detection limit. Expression was determined with quantitative real-time PCR, mean with S.D. of three determinations are displayed. c-e Vangl2 overexpression reduced the frequency of Tuj1 positive C17.2 cells (C, compare i and iii). For quantification 68 control and 84 Vangl2 cells were scored between 1 (no Tuj1 labeling) and 5 (very high labeling). All control transfected cells were Tuj1 positive (score 2–5). Contrary, more than half (56 %) of the Vangl2 transfected cells did not display any Tuj1 label (score 1) d. Vangl2 increased active β-catenin in the nucleus of C17.2 cells (C, compare v and vii). Active β-catenin was found in the nuclei of 33 % of the enhanced green fluorescent protein and 48 % of the HA-Vangl2 (t-test, P = 0.0037) e, mean with S.D. are shown. Scale bar: 10 μM. f Vangl2 knockdown in C17.2 cells induced neural outgrowth, morphology consistent with increased differentiation compared to control transfected cells. Scale bar: 10 μM. g The transcriptional activity of β-catenin measured as TOPflash luciferase activity was significantly reduced after Vangl2 knockdown in C17.2 cells (t-test, P = 0.0092). **P < 0.01, ***P < 0.001