Fig. 2

Ogt deficiency induces inflammatory response of astrocytes. a Representative images of GFAP and IL-1β immunostaining with cultured adult Ctrl and cKO astrocytes in vitro. Scale bar, 50 μm. b, c qRT-PCR results showed that mRNA levels of IL-1β (b) and TNF-α (c) increased in cKO astrocytes. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. d–f WB assay (d) and quantification results showed that protein levels of IL-1β (e) and TNF-α (f) significantly increased in adult cKO astrocytes in vitro. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. g Representative images of GFAP and IL-1β immunostaining in the hippocampus of brains of Ctrl and cKO mice. Scale bar, 20 μm. h, i qRT-PCR results showed that mRNA levels of IL-1β (h) and TNF-α (i) 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. j–l WB assay (j) and quantification results showed that the IL-1β (k) and TNF-α (l) protein levels significantly 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. m, n ELISA assay results show that the levels of IL-1β (m) and TNF-α (n) increased in the hippocampal tissue supernatants of cKO mice compared to Ctrl mice. n = 6 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. o–q WB assay (o) and quantification results showed that the treatment with Ogt inhibitor (Ogt-I) OSMI-4 reduced the level of O-GlcNAcylation in adult astrocytes (p), but increased the level of GFAP (q) compared to Ctrl group. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. r Representative images of GFAP and IL-1β immunostaining of adult astrocytes treated with PBS (Ctrl) and Ogt-I (20 μM for 72 h), respectively. Scale bar, 50 μm. s–u WB assay (s) and quantification results show that the treatment with Ogt inhibitor (Ogt-I) OSMI-4 significantly increased the levels of IL-1β (t) and TNF-α (u) of adult astrocytes in vitro. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test