Fig. 3

Increased binding of H3K9me3 to the GFAP, S100B, and GDNF promoter in the hyperglutamate environment. Histone demethylase inhibitor D-2HG downregulates the expression of characteristic glial cell proteins for BMSCs. A–C Association of H3K9me3 with GFAP, S100B, and GDNF was assessed by chromatin immunoprecipitation (ChIP) assays. DNA was immunoprecipitated with the specific antibody H3K9me3. Bars represent the relative levels of the PCR product of the GDNF (Control vs. Glu: 1 ± 0.10 vs. 1.81 ± 0.34, P < 0.05), S100B (Control vs. Glu: 1 ± 0.05 vs. 1.62 ± 0.27, P < 0.05) and GFAP (Control vs. Glu: 1 ± 0.17 vs. 1.54 ± 0.21, P < 0.05) promoter region’s association with H3K9me3. Non-immunized serum (IgG) was used as a control. Each qPCR assay was repeated two times. Values are the mean ± SD from three technical replicates. BMSCs were incubated with D-2HG (2 mM) or Glu (2 mM) for 24 h. D and E Representative immunoblot bands and histogram of relative expression for the H3K9me3 (Control:1, Glu: 0.31 ± 0.04, D-2HG: 1.62 ± 0.18, Glu + D-2HG: 1.76 ± 0.18), H3K9me1 (Control:1, Glu: 1.71 ± 0.34, D-2HG: 0.56 ± 0.19, Glu + D-2HG: 0.28 ± 0.10), H3K27me3 (Control:1, Glu: 0.50 ± 0.06, D-2HG: 1.67 ± 0.13, Glu + D-2HG: 1.79 ± 0.17), and H3K27me1 (Control:1, Glu: 1.95 ± 0.19, D-2HG: 0.66 ± 0.09, Glu + D-2HG: 0.56 ± 0.08) proteins. H3 was used as a loading control. F and G Representative immunoblot bands and histogram of relative expression for the GFAP (Control:1, Glu: 1.39 ± 0.11, D-2HG: 0.62 ± 0.05, Glu + D-2HG: 0.69 ± 0.08), S100B (Control:1, Glu:1.38 ± 0.19, D-2HG: 0.76 ± 0.11, Glu + D-2HG: 0.66 ± 0.12), and GDNF (Control:1, Glu: 1.49 ± 0.23, D-2HG: 0.59 ± 0.05, Glu + D-2HG: 0.50 ± 0.16) proteins. GAPDH was used as a loading control. Glu: glutamate; D-2HG: D-2-hydroxyglutaric acid; ChIP: chromatin immunoprecipitation. These results are representative of at least three times independent experiments. *P < 0.05, **P < 0.01