Fig 2: E4 microglia exhibit increased interferon signaling, increased eIF2 signaling, and decreased protein translation at baseline. a) Expression of several genes from the interferon a/ß and interferon ? signaling pathways (Fig. 4d) were measured from cell lysates of E3 and E4 microglia using qRT-PCR. E4 microglia exhibited an increase in the expression of several of these genes compared to E3 microglia; unpaired Welch’s t-test followed by Benjamini-Hochberg (BH) correction for multiple comparisons, Ifnar1 avg. fold change (FC) ± SEM = 1.1 ± 0.4, adj. p = 1.0; Ifnar2 FC = 3.0 ± 0.6, adj. p = 0.02; Irf9 FC = 1.1 ± 0.2, adj.p = 1.0; Irf2 FC = 0.31 ± 0.04, adj. p = 0.005; Irf5 FC = 1.4 ± 0.1, adj. p = 0.08, Irf8 FC = 4.8 ± 0.6, adj. p = 0.002; Stat1 FC = 2.8 ± 0.40, adj. p = 0.06; Stat2 avg. FC = 7.8 ± 0.85, adj. p = 0.0003; Oas1g FC = 1.6 ± 0.2, adj.p = 0.06; Oas2 avg. FC = 5.1 ± 0.60, adj. p = 0.0004; Oas3 avg. FC = 3.8 ± 0.29, adj p = 0.0004; Irf3 FC = 1.0 ± 0.08, adj. p = 1.0; Irf7 avg. FC = 11.7 ± 1.1, adj. p = 5.7 × 10-05; Il1ß FC = 1.0 ± 0.1, adj. p = 1.0; Tnfa avg. FC = 1.90 ± 0.07, adj. p = 0.02; Cxcl1 FC = 1.1 ± 0.2, adj. p = 1.0; Ccl2 avg. FC = 4.5 ± 0.41, adj. p = 0.0003; Il6 avg. FC = 8.8 ± 1.4, adj. p = 0.003, Ccl5 FC = 7.9 ± 4.0, adj. p = 0.06; Cxcl10 avg. FC = 213.5 ± 12.3, adj. p = 3.1 × 10-07; Ikke avg. FC = 1.9 ± 0.1, adj. p = 0.01; Cxcl9: 669.5 ± 187.9, adj. p = 0.004; Jak1 FC = 0.7 ± 0.1, adj. p = 0.3; Irf1 avg. FC = 50.3 ± 3.1, adj. p = 6.05 × 10-05. b) Levels of select secreted cytokines/chemokines and other inflammatory mediators were measured in the conditioned media from E3 and E4 microglial cultures using a Cytokine Proteome Profiler array kit. E4 microglia showed significantly increased levels of specific cytokines/chemokines compared to E3: unpaired Student’s t-test followed by BH correction for multiple comparisons, IL1a t(8) = 4.7, adj. p = 0.01, d = 3.0 CI95% [1.0, 4.8]; CCL2 t(8) = 4.2, adj. p = 0.02, d = 2.7 CI95% [0.8, 4.4]; CCL12 t(8) = 4.1, adj. p = 0.02, d = 2.6 CI95% [0.8, 4.3]; CCL3 t(8) = 5.8, adj. p = 0.007, d = 3.6 CI95% [1.5, 5.7]; CCL4 t(8) = 5.3, adj. p = 0.007, d = 3.4 CI95% [1.3, 5.4]; CXCL2 t(8) = 5.3, adj. p = 0.007, d = 3.4, CI95% [1.3, 5.4]; CCL17 t(8) = 1.5, adj. p = 0.8, d = 1.0 CI95% [-0.4, 2.3]; TNFa t(8) = 5.3, adj. p = 0.007, d = 3.3 CI95% [1.2, 5.3], n = 5 independent experiments. c) Levels of secreted interferons in the supernatant of E3 and E4 microglia cultures were measured using ELISA. E4 microglia showed no significant change in interferon-a/ß; t(3.8) = -0.6, p = 0.6, d = -0.5 CI95% [-1.8, 1.0], but exhibited a significant increase in interferon-? t(2) = 10.8, p = 0.008, d = 8.8 CI95% [2.8, 14.9] relative to E3 microglia. d) Protein synthesis was measured in E3 and E4 microglia using the SUnSET assay, which detects levels of puromycin incorporated into newly synthesized proteins. A representative western blot is shown. M indicates the molecular weight ladder and both short exposure (top) and longer exposure (bottom) are displayed. e) Original non-over exposed western blot (top) was quantified and shows decreased puromycin incorporation in E4 microglia at 1 hour compared to E3, t(3.0) = -8.6, p = 0.003, d = -6.1 CI95% [-10.9, -1.4]. f) Representative Western blots showing the protein levels of both total and phosphorylated eIF2a, PRK, PERK, and GCN2 protein. g) Quantification of western blots shows E4 microglia exhibit a significant increase in the ratio of phosphorylated to total eIF2, t(4.8) = 4.6, p = 0.007, d = 3.2 CI95% [0.8, 5.6] and PKR; t(3.0) = 8.6, p = 0.04, d = 2.5 CI95% [0.1, 4.9] compared to E3 microglia. The ratio of p-PERK to total PERK was increased but did not reach statistical significance, t(3.0) = 2.7, p = 0.07, d = 1.9 CI95% [-0.2, 3.9] while the percentage of GCN2 that was phosphorylated remained unchanged, t(3.3) = 0.1, p = 0.92, d = 0.1 CI95% [-1.3, 1.5]. a, c, e, g) Each dot corresponds to an independent experiment (n = 4/group). Black horizontal bars indicate the mean and error bars represent SE. c, e,g) Unpaired Welch’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: UPR-Reactive Astrocytes Have an Altered SecretomeThe proteome of conditioned media from vehicle-, Tg-, or Tg and GSK2606414-treated astrocytes was analyzed by LC/MS.(A) Heatmap showing normalized spectral counts of the 25 most abundant proteins detected in the conditioned media of vehicle-treated astrocytes. 10 of the 25 proteins showed reduced spectral counts on Tg treatment. These changes were largely reversed by PERK inhibition.(B) Pie chart showing the percentage of proteins that exhibited an increase, decrease, or no change in spectral counts on Tg treatment.(C) Bubble plots showing KEGG and GO functional analysis of the proteins that displayed reduced spectral counts on Tg treatment, as determined using DAVID. Terms relating to the extracellular matrix and cell adhesion were significantly enriched upon PERK activation (p < 0.0001). Size of bubble represents protein count. LC/MS was performed on conditioned media from 3 biological replicates.

Fig 4: 4-PBA and TUDCA suppress post-IRI ER stress in kidney tubules. C57Bl/6 mice (male, 8–10 week old) were subjected to 30 min of unilateral ischemia of left kidney followed by reperfusion for 7 or 14 days. 4-PBA at 20 mg/kg/day, TUDCA at 250 mg/kg/day, or normal saline (NS) was given from day 3 after uIR. Then kidney tissues were collected for biochemical and histological analyses. (A) Representative images of immunohistochemical staining of BiP. Bar = 200 µm. (B) Quantitative analysis of BiP staining. (C) Representative immunoblots of p-PERK, PERK, BiP, CHOP, and GAPDH. GAPDH was used as a loading control. (D) Quantitative analysis of immunoblot signals of p-PERK, BiP and CHOP. For densitometric analysis, the protein level of sham group was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control group to indicate their protein fold changes. (E) Representative images of immunohistochemical staining of CHOP. Arrows point to positive CHOP staining. Bar = 200 µm. (F) Quantitative analysis of CHOP staining. Data are expressed as mean ± SEM. n = 4. *p < 0.05;**p < 0.01;***p < 0.001; ns, not significant.

Fig 5: Protein expression of the mediators of the PERK-UPR pathway. Western blot analysis revealed no significant differences in the expression of (A) p-PERK, (B) p-eIF2a, (C) CREB2, or (D) GADD34 between injury or treatment groups. Mean ± SEM. CREB2, cAMP response element-binding protein-2; eIF2a, eukaryotic translation initiation factor a; GADD34, growth arrest and DNA-damage-inducible 34; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSK, GSK2606414; p-eIF2a, phosphorylated eIF2a; PERK, protein kinase R-like endoplasmic reticulum kinase; p-PERK, phosphorylated PERK; SEM, standard error of the mean; TBI, traumatic brain injury; UPR, unfolded protein response; VEH, vehicle.