Fig. 1

Ogt deficiency leads to activation of astrocytes in vitro and in vivo. a, b Representative images of GFAP immunostaining (a) and quantification results showed that Ogt deficiency increased the average area of adult astrocytes (b). Scale bar, 50 μm. n = 10 astrocytes were picked up per animal and 30 cells in total were analyzed per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. c, d Western blot (WB) assay (c) and quantification results (d) showed that the protein level of GFAP significantly increased in cKO astrocytes compared with Ctrl astrocytes. Astrocytes isolated from 2 to 3 adult mice were pooled together and regarded as n = 1 in the present study. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. All the original images of western blot assays can be found in Additional file 10: Fig. S10. e–g qRT-PCR results show that mRNA levels of pan reactive (e) and A1 specific (f) markers increased in Ogt deficient adult astrocytes, and A2 specific markers decreased (g). n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. h Representative images of GFAP and Iba1 immunostaining. Scale bar, 100 μm. The right panels showed the higher magnification of the area marked with white frame in left panel images. Scale bar (right panels, 20 μm). i–l Quantification results showed that the level of GFAP fluorescence intensity (i) and the number of GFAP⁺ cells (j) significantly increased in the hippocampus region of cKO mice compared to Ctrl mice. n = 4 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. k–n three-dimension (3D) analysis of astrocytic morphology showed the increase of the total length (k), the number of processes (l), the process volume (m) and total process area (n) of astrocytes from the hippocampus of adult cKO mice compared with Ctrl mice. n = 10 astrocytes were picked up per animal and 40 cells in total were analyzed per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. o 3D Sholl analysis showed the increased process arbor complexity of astrocytes in the hippocampus of cKO mice compared with Ctrl mice. n = 10 astrocytes were picked up per animal and 40 cells in total were analyzed per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA followed by Sidak’s multiple comparisons test, F(_{1, 5304)} = 2258. p qRT-PCR results showed that mRNA level of GFAP increased in the hippocampal tissues of cKO mice compared to Ctrl mice. n = 5 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. q–s WB assay (q) and quantification results showed that the protein level of GFAP (r) increased in the hippocampal tissues of cKO mice compared to Ctrl mice, but the level of Iba1 (s) showed no difference between Ctrl and cKO mice. n = 6 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test