Fig 2: Western blot analysis of protein expression levels of key enzymes and transporters in glutamatergic and monoaminergic pathways in the EC of mice after CRS exposure. ß-actin and GAPDH were used to normalize the expression levels of proteins in control group and CRS group. (A) The protein expression levels of SERT, TH, AADC, MAOA and VMAT2 in monoaminergic pathway were examined by western blott (* P<0.05, n=6-7 mice per group). (B) The protein expression levels of GAD1, EAATT2, GLUL and SNAT1 in glutamatergic pathway were analyzed in both groups. (* P<0.05; n=6-7 mice per group).

Fig 3: RT-qPCR validation of mRNA expression levels of key enzymes and transporters in glutamatergic and monoaminergic pathways in the EC of mice after CRS exposure. ß-actin and GAPDH were used to normalize the expression levels of genes in control group and CRS group. (A) The mRNA expression levels of SERT, AADC, MAOA, TPH1, TPH2, NET and COMT in monoaminergic pathway were examined by RT-qPCR. (B) The mRNA expression levels of GAD1, EAAT2, GLUL, SNAT1, GLS and VGLUT1 in glutamatergic pathway were tested in both groups through RT-qPCR. (* P<0.05, ** P<0.01, *** P<0.001; n=6-8 mice per group).

Fig 4: Validation of proteomic results and expression levels analysis in MUT-Rescue cells. According to the results of the proteomic LFQ, the downregulation of VIM, SDC2, EXT2, FN1, and the upregulation of GLUL were confirmed in MUT-KO cells by WB. The expression levels of these proteins were also measured in MUT-Rescue cells. (a) The panel reports blot scans of X-ray films for each tested protein and their loading controls. (b) Densitometry analysis results are reported as the mean ± SD of three biological replicates each normalized to the amount of a-tubulin, ß-actin, or GAPDH proteins. A one-way two-tail t-test was used to calculate the statistical significance of differences between the three groups (p value). *: p < 0.05; **: p < 0.01; ***: p < 0.005; NS: Not significant (p > 0.05).

Fig 5: Optic nerve head astrocyte characterization. (A) Isolated optic nerve head (ONH) astrocytes were immunolabeled using antibodies against GFAP (green), ß-tubulin (red, upper panels), and Iba1 (red, lower panels) to determine the purity of the cell cultures. DAPI was used to stain cell nuclei (blue). Neither ß-tubulin nor Iba1 was detected in the cultures. Scale bar = 10 µm. (B) Sections of retina with ONH and optic nerve attached served as positive controls for the immunolabeling in (A). Both ß-tubulin and Iba1 were observed in the mouse ONH and optic nerve. Scale bar = 25 µm. (C) Quantification of ß-tubulin and Iba1 protein in the optic nerve head (ONH) and isolated ONH astrocytes using capillary electrophoresis. Neither ß-tubulin nor Iba1 was detected in the ONH astrocyte cultures (square symbols); n = 3 biological replicates per group. (D) Quantitative PCR for a number of glial transcripts and transcripts for retinal ganglion cells (Rbpms), microglia (Iba1), and oligodendrocytes (Mbp) was used to further characterize the ONH astrocytes. Rbpms, Iba1, and Mbp were not detected in mRNA isolated from control or stretched ONH astrocytes (ND). There were no differences in transcripts for Gfap, Slc1a3 (glutamate-aspartate transporter), Glud1 (glutamate dehydrogenase-1), and Glul (glutamine synthetase). There were significant differences in the Gja1 transcript levels, with stretched ONH astrocytes showing greater fold change as compared to control (*p = 0.0152, n = 3). Gja1 encodes Connexin-43; n = 3 biological replicates per group. (E) GFAP protein, increased when astrocytes undergo hypertrophic reactivity, did not statistically differ between control and stretched ONH astrocytes; n = 3 biological replicates per group. (F) S100ß protein, also upregulated in reactive astrocytes, did not statistically differ between control and stretched ONH astrocytes; n = 3 biological replicates per group.