Additional file 2 of Glutamate regulates gliosis of BMSCs to promote ENS regeneration through α-KG and H3K9/H3K27 demethylation

Additional file 2: Fig. S1. The relative glutamate concentration, the expression levels of inflammatory cytokines (IL-1β/TNF-α/IL-6) and anti-inflammatory cytokines (IL-13/IL-10/IL-4) of gastric tissue in different groups of mice. A and B Mouse stomach tissue presents a high-glutamate microenvironment after nerve injury, and BMSCs transplantation reduced the glutamate concentration. C–H The transcripts of inflammatory and anti-inflammatory cytokines were determined by RT-PCR assay. The inflammatory cytokines (TNF-α/IL-6) and anti-inflammatory (IL-13/IL-10/IL-4) were increased in ENS injury mice compared with control, and BMSCs transplantation reduced the level of inflammatory cytokines (TNF-α/IL-6), increased the level of anti-inflammatory (IL-13). BAC: benzalkonium chloride-treated mice; BAC + BMSCs (NC-Glud1): BAC mice transplanted with BMSCs-NC; BAC + BMSCs (OE-Glud1): BAC mice transplanted with BMSCs-OE; ENS: enteric nervous system. Results were expressed as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001, NS: no significance. Fig. S2. Glutamate increases the expression of glial cell characteristic protein for BMSCs. Different concentrations of glutamate (0, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 4 mM) were incubated with BMSCs for 24 or 48 h. A–D Representative immunoblot bands and histogram of relative expression for the GFAP and GDNF proteins. GAPDH was used as a loading control. E and F Fluorescent label-CFSE proliferation detection of BMSCs and statistical analysis. Glu: Glutamate; CFSE: carboxyfluorescein diacetate succinimidyl ester. These results are representative of at least three times independent experiments. *P < 0.05, **P < 0.01, NS: no significance. Fig. S3. The GABA receptors of BMSCs is activated under glutamate intervention. A and B The transcripts of GABARA and GABARB genes of BMSCs treated with glutamate were determined by a RT-PCR assay, and the GABARA genes of BMSCs is activated with glutamate intervention. ***P < 0.001, NS: no significance. Fig. S4. The mesenchymal genes of BMSCs is activated under glutamate intervention. A and B The transcripts of mesenchymal genes (Snail and Twist) in BMSCs treated with glutamate were determined by a RT-PCR assay. C–E The transcripts of genes involved in cell migration (MMP2, MMP9 and CXCR4) of BMSCs treated with glutamate were determined by a RT-PCR assay, and CXCR 4 of BMSCs is activated under glutamate intervention (P < 0.05). *P < 0.05, **P < 0.01, ****P < 0.0001, NS: no significance. Fig. S5. The intracellular α-KG, succinate concentration and α-kg/succinate ratio of BMSCs. BMSCs were incubated with D-2HG (2 mM) or Glu (2 mM) for 24 h. The intracellular α-KG content and the ratio of α-KG/succinate of BMSCs were significantly increased under glutamate intervention, and D-2HG reversed this change. Glu: Glutamate; D-2HG: D-2-hydroxyglutaric acid. These results are representative of at least three times independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance. Fig. S6. Knockdown of Glud1 derived from the BMSCs could resulted in decreased expression of the characteristic glial cell proteins. A Transfection of recombinant lentiviral vectors into BMSCs and resultant changes in Glud1 expression. Fluorescence microscope patterns of BMSCs-NC, BMSCs-KD and BMSCs-OE. B and C Representative immunoblot bands and histogram of relative expression Glud1 protein in BMSCs-NC, BMSCs-KD and BMSCs-OE. D–F The intracellular α-KG, succinate concentration and α-kg/succinate ratio of BMSCs. The intracellular α-KG content and the ratio of α-KG/succinate of BMSCs were significantly increased under glutamate intervention, and knockdown of Glud1 derived from the BMSCs reversed this change. G and H Representative immunoblot bands and histogram of relative expression of for the GFAP, S100B, and GDNF in each group. GAPDH was used as a loading control. Glu: Glutamate; Glud1: Glutamate Dehydrogenase 1; OE: overexpression; KD: knockdown; NC: negative control. These results are representative of at least three times independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: no significance. Fig. S7. The genes associated with glial cell properties of BMSCs is activated under glutamate intervention and in OE-Glud1 BMSCs. A and B The transcripts of neuronal genes (PGP9.5 and β-tubulin) of BMSCs in different groups were determined by a RT-PCR assay. C–E The transcripts of glial related genes (GFAP, GDNF, S100B) of BMSCs in different groups were determined by a RT-PCR assay. F and G The transcripts of neurotrophic factor genes (BDNF, NGF) of BMSCs in different groups were determined by a RT-PCR assay. H The transcripts of cell proliferation-related genes (PCNA) of BMSCs in different groups were determined by a RT-PCR assay. I The transcripts of cell apoptosis related genes (Caspase-1) of BMSCs in different groups were determined by a RT-PCR assay. *P < 0.05, **P < 0.01, NS: no significance. Fig. S8. The genes associated with oxidative stress of BMSCs is activated under glutamate intervention and in OE-Glud1 BMSCs. A–C The transcripts of oxidative stress related genes (COX5a, SOD2, GPX4) in BMSCs treated with glutamate were determined by a RT-PCR assay. COX5a/SOD2 of BMSCs is activated under glutamate intervention and in OE-Glud1 BMSCs. *P < 0.05, **P < 0.01, NS: no significance.