Fig. 8

The intracellular localization of β-catenin, the major WNT signaling pathway mediator, is regulated by primary cilia. A-B The active (non-phosphorylated) form of β-catenin is significantly upregulated in an RFX2 -/- background as compared to WT neurons. This upregulation is particularly strong on d2 and d3 of neuron differentiation, the two time points with the highest percentage of ciliation (cf. Fig. 2A). Quantification of expression values (A) was carried out from western blot analysis (B) relative to the housekeeping protein GAPDH (loading control). C-D Active β-catenin immunocytochemistry staining of WT and RFX2 -/- neurons highlights differences between genotypes in intracellular protein localization: note the more prominent nuclear localization in the RFX2 -/- background and in non-ciliated WT neurons as compared to ciliated WT neurons. Circles define nuclei and dashed lines define the cell body; the ciliary marker is ARL13B (green). E–F Quantification of active β-catenin localization based on the ratio of immunofluorescence intensity between the cytoplasm and the nucleus on d1, d2 and d3 of LUHMES neuron differentiation. In WT, ciliated neurons (n = 39–167) more strongly promote β-catenin degradation in the cytoplasm, resulting in diminished translocation to the nucleus of the active form (lower ratio), as compared to non-ciliated neurons (n = 44–95) (higher ratio) (E). In an RFX2 -/- background, ciliated (n = 16–77) and non-ciliated neurons (n = 28–57) show no differences in regulating β-catenin intracellular localization (equal ratios) (F). Mean values are shown ± s.e.m. (normalized to GAPDH) (A) and ± s.d. (E–F). The results are from two independent experiments with a minimum of two technical replicates each. We conducted regular two-way ANOVA analyses (not repeated measures) with multiple comparisons (Bonferroni’s test) between groups. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001