Figure 1. CK1δ Expression in Postnatal GCPs and Control of GCP Proliferation in vitro and ex vivo.

(A) Cerebellar sections from P8 pups were stained with antibodies against CK1δ (green) or calbindin (red) and DAPI (blue). (B) CK1α , CK1δ, and CK1ε mRNA was amplified by qRT-PCR, and fold-change in gene expression in postnatal mouse cerebellum was determined by normalizing to GAPDH values relative to control. (C) GCPs were incubated for 24 h with increasing concentrations of SR-653234 or SR-1277, and the amount of proliferation was determined by ³H-thymidine incorporation. Results were plotted relative to that seen in the DMSO control. (D) GCPs were treated with 100 nM SR-653234 or SR-1277 for 24 h, and then Sytox and Hoechst staining was performed (NS, not significant, as determined by one-way ANOVA and Dunnett multiple comparisions test). (E) D4476 (20 μM) reduces GCP proliferation, and (F) SR-653234 (100 nM) and SR-1277 (100 nM) increase the percentage of GCPs in the S or G2/M phase. GCPs were treated for 24 h with the indicated compounds or DMSO, and the proportion of cells in each cell cycle phase was determined by PI-FACS. (G) Organotypic cerebellar slices were treated with SR-1277 (100 nM), SR-653234 (100 nM), D4476 (20 μM), or DMSO for 1 h, after which EdU was added to the media for 20 h. Slices were stained with EdU (red) and the nuclear marker DAPI (blue). (H) Quantification of (G). Results are shown as the average values of three independent experiments and are represented as the mean and standard error of the mean (± SEM) (*p <0.05, **p <0.001, ***p <0.001, ****p <0.0001).