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. 2023 Jun 21;43(25):4559–4579. doi: 10.1523/JNEUROSCI.1962-22.2023

Figure 2.

Figure 2.

cKO mice display cerebellar atrophy and the reduced Ogt and O-GlcNAcylation. A, Representative brain images of adult Ctrl and cKO mice. The dashed lines outline the area of cerebellum. B, Weight of the cerebellum of adult Ctrl and cKO mice. n = 6 mice per genotype. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001. C, Representative images of NeuN and Calbindin immunostaining in the cerebellum of adult Ctrl and cKO mice. Scale bar, 500 μm. D, E, Quantification of the cerebellum area (D) and cerebellar granule cell layer (CGL) area (E) of adult Ctrl and cKO mice. n = 3 mice for each group, and 6 sections from each animal. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001. F, Representative images of NeuN and Ogt immunostaining in the cerebellum of adult Ctrl and cKO mice. Scale bars, 50 μm (left, lower magnification) and 10 μm (right, higher magnification). G, Quantification of the fluorescence intensity of Ogt in (F). n = 3 mice for each group, and 3 sections from each animal. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001. H, Representative images of O-GlcNAcylation in the granular cell layer (GCL) of cerebellum of adult Ctrl and cKO mice. Scale bar, 20 μm. I, Quantification of the relative fluorescence intensity of O-GlcNAcylation in H. n = 3 mice for each group, and 3 sections from each animal. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001. J–L, WB assays (J) and quantification results of Ogt (K) and O-GlcNAcylation (L) levels in the cerebellum of adult Ctrl and cKO mice, respectively. n = 3 independent experiments for each group. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001. M, qRT-PCR assay of Ogt mRNA expression in the cerebellum of adult Ctrl and cKO mice. n = 3 independent experiments for each group. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001. N–P, Representative images of Tbr2 and Ogt immunostaining (N) and quantification of the fluorescence intensity of Ogt in Tbr2+ cells (O) and the density of Tbr2+ UBCs (P) in the lobule VI of cerebellum of adult Ctrl and cKO mice. n = 3 mice for each group, 3 sections from each animal and 6 visual fields per section. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001, ns, not significant. Scale bar, 20 μm. Tbr2, a marker for unipolar brush cells. Q–S, Representative images of Ogt and SMI32 (left), and O-GlcNAcylation (right; Q) and quantification of the fluorescence intensity of Ogt (R) and O-GlcNAcylation (S) in the deep cerebellum nuclei (DCN) of adult Ctrl and cKO mice. n = 3 mice for each group, and 3 sections from each animal. Data are presented as mean ± SEM. Unpaired Student's t test, *p < 0.05, **p < 0.001, ***p < 0.0001, ns, not significant. SMI32, a marker for deep cerebellum nuclei. Scale bar, 50 μm.