Figure 4.

Lack of IP6K2 and 4.1N, highly expressed granule cells proteins, leads to diminished proliferation of cerebellar cells. A, Immunofluorescent staining of wild-type mouse brain cerebellar sections showed localization of IP6K2 and 4.1N in granule cells of the cerebellum. B, Western blots for calbindin and calretenin, respective markers for Purkinje and granule cells, showed reduced expression of both proteins in mouse brain lysates lacking IP6K2. C, Histological comparison through H&E staining of WT and IP6K2 KO brain (IP6K2-KO; 8-week-old) sections showed substantial decreases in the molecular layer compared with the WT. GCL, Granule cell layer; PCL, Purkinje cell layer; ML, molecular layer. D, H&E-stained sections of IP6K2-KO mouse also showed substantially fewer granule cells in the cerebellar region of mutant mice compared with WT. E, F, Mice injected with EdU showed fewer proliferating cells (marked with white arrow heads) in IP6K2-KO mice compared with the WT. G, XTT assay performed with knockdown of K2 as well as K2 lacking binding site for 4.1N (202–261) in N2A cells also revealed lower cell viability compared with control shRNA. Scale bar, 50 μm. The data are presented as the mean ± SD: ***p < 0.001, **p < 0.01, analyzed by one-way ANOVA. The H&E staining of wild type (WT) and IP6K2-Knockout (KO) sagittal brain sections (A), Hippocampus (B) and Cerebellum (C) is shown in Figure 4-1.