Fig 2: Significant increase of CHOP expression in the GCL 1 day after AION. (A) Increased CHOP+Brn3A+ cells in the GCL 1 day after AION. (B) Representative images showing increase in the number of CHOP+ cells in the GCL and increase in CHOP immunoreactivity. (C) Bar graph showing 10-fold significant increase in CHOP+DAPI+ cells in the GCL 1 day after AION (*P = 0.03, Mann-Whitney U test). (D) Significant increase in the intensity of CHOP immunoreactivity in the GCL 1 day after AION (*P = 0.03, Mann-Whitney U test). Scale bar: 25 µm. GCL, ganglion cell layer.

Fig 3: Interference with the endonuclease activity of inositol-requiring protein 1 (IRE1) attenuates induction of type I interferon (IFN) response after proteasome impairment. (A) Picture showing spliced variant of X-box-binding protein 1 (XBP1), detected by PCR and visualized on an agarose gel electrophoresis. (B) Immunoblotting depicting expression of type I IFN response drivers in BV-2 cells treated with 50 nM of bortezomib. Cells were pretreated for 2 h with 100 µM of the 4µ8C inhibitor or with dimethyl sulfoxide (DMSO) as a control. Quantitative reverse transcription PCR (RT-PCR) analysis of (C) Ifnß1, (D) Cxcl-10, and (E) Il-6 mRNA levels from whole RNA extracts of BV-2 cells. Mean ± SEM (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-sided t-test. (F) Expression of C/EBP homologous protein 10 (CHOP) and activating transcription factor 6 (ATF6) transcription factors in BV-2 after the IRE1 inhibition and treatment with 50 nM bortezomib.

Fig 4: No change in GRP78 in the anterior optic nerve cells 1 day after AION. (A) No change in GRP78+Olig2+ cells in the anterior optic nerve 1 day after AION. (B) Representative images and (C) bar graph showing no change in CHOP+DAPI+ cells in the optic nerve 1 day after AION. (D) Bar graph showing no change in the intensity of GRP78 immunoreactivity in the anterior optic nerve 1 day after AION (n = 4–5, P = 0.773, Mann-Whitney U test). Scale bar: 25 µm.

Fig 5: Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (Mø), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 µm. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 µM of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2a, and total- eIF-2a. ß-Actin was used to determine equal loading of samples.