Fig 2: CNβ null mice exhibit larger infarct size after photo thrombotic stroke as compared to CNβ null mice with inhibition of PERK activity. PERK inhibitor (PI) or DMSO (Con) was intraperitoneally injected into CNβ wildtype (β+/+) and null (β−/−) mice 12 h before stroke. (A) Homogenized brain lysates of mice at 12 h post-injections were analyzed for T-PERK, P-PERK, T-eIF2α and P-eIF2α by immunoblots. (B) Quantification of immunoblots in (A) (n = 3, mean ± SEM, *p < 0.05 by unpaired two-tailed Student’s t-test). (C) Rose bengal (RB) dye-induced photothrombotic model. Confocal images show that blood vessel lumens were filled with RB dye. Region marked by dashed box was irradiated with 543 nm laser light. After approximately 5 min, a thrombotic clot was formed with the absence of blood flow. Scale bar: 20 µm. (D) Tetrazolium chloride (TTC) staining of serial coronal sections 24 h post-photothrombotic stroke on β+/+ and β−/− mice with DMSO (Con) or PI injection. Scale bar: 5 mm. (E) Quantification of infarct volume 24 h post-stroke in (D) (n = 5, mean ± SEM, *p < 0.05 by one-way ANOVA).

Fig 3: CNβ induces PERK phosphorylation independent of its phosphatase activity. (A, B) Primary astrocytes cultured from wildtype mice were transduced with pUltra lentivirus containing empty vector (Lenti-GFP Con) or Lenti-GFP (CNβ) and stained for CNβ (red). Nuclei were stained with DAPI (blue). Presence of virus is indicated by GFP (green). (C–F ) Infected astrocytes were exposed to DMSO (Veh) or 40 µM of Quercetin (QC) for 20 min. Immuno-cytochemistry was carried out for P-PERK (red) and DAPI (blue). Images were sequentially acquired. Scale bar: 20 µm. (G) Ratio of fluorescence intensity (CNβ/GFP) in (A, B) from three independent experiments in which at least 10 cells were quantified per experiment (mean ± SEM, ***p < 0.001 by unpaired two-tailed Student’s t-test). (H) Ratio of fluorescence intensity (P-PERK/GFP) in (C–F) from three independent experiments in which at least 10 cells were quantified per experiment (mean ± SEM, ***p < 0.001 by one-way ANOVA).

Fig 4: CNβ physically interacts with PERK and promotes PERK phosphorylation and oligomerization. (A) C8D1A type I astrocytes were treated with vehicle (DMSO; Con) or 1 µM of thapsigargin (Tg) for 1 h. Cell lysates were analyzed for total (T) and phosphorylated (P) PERK and eIF2α using immunoblots. (B) Densitometry histograms after normalization to T-PERK or T-eIF2α, respectively (n = 3, mean ± SEM, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student’s t-test). (C) Primary mouse astrocytes were treated as indicated in (A) and then cross-linked with disuccinimidyl suberate (DSS) for 30 min. Immunoprecipitation (IP) was performed with anti-CNβ antibody and subsequent blots were probed with anti-PERK antibody. (D) Quantification of P-PERK/T-PERK densitometric ratio in (C) (n = 3, mean ± SEM, *p < 0.05 by unpaired two-tailed Student’s t test). (E) GST pull-down assay with either 8 nM of CNα or CNβ. The proteins were incubated with glutathione sepharose 4B for 1 h, resolved on a 12% SDS-polyacrylamide gel and probed with an anti-calcineurin PAN-A antibody. CN pull-down levels are shown for GST alone and GST-cPERK. (F) Densitometric histogram of (E) (n = 3, mean ± SEM, **p < 0.01 by unpaired two-tailed Student’s t test). (G) GST-cPERK was added to all reaction mixtures along with [g32P] ATP in the absence or the presence of either of 0.043 mM of CNα or CNβ. Reaction mixtures were run on SDS-PAGE and visualized by autoradiography. (H) Quantification of cPERK auto-phosphorylation density (ne = 5, mean ± SEM, *p < 0.05 by one-way ANOVA). (I) Recombinant His-CNβ and GST-cPERK were incubated at the concentrations indicated, in the presence of 0.3 mM of DSS cross-linker for 30 min at room temperature. The ensuing protein complexes were run on SDS-PAGE and detected by immunoblotting using an anti-PERK antibody. (J) Recombinant His-CNβ (1.2 mM) and GST-cPERK (0.01 mM) were incubated in the presence of 0.3 mM of DSS for 30 min at room temperature. The protein complexes were run on SDS-PAGE and detected by immunoblotting using antibodies against CNβ and PERK.

Fig 5: Lentiviral expression of CNβ increases cell viability of wildtype but not PERK-null astrocytes. (A, C, F, H) Astrocytes cultured from Perk+/+ and Perk−/− mice were infected with lentivirus pUltra lenti-GFP or pUltra lenti-GFP (CNβ) (green). Cells in (B, D) were exposed to tBuOOH for 2 h and cells in (G, I) were exposed to OGD for 1 h. Cells in (K) were at rest. Nuclei were stained using Hoechst 33342 (blue). Cells with GFP expression were viable cells. Scale bar: 80 µm. (E, J) Quantification of percent live cells in lentivirus-infected astrocytes in (B, D, G, I) from three independent experiments in which 5 random fields were counted (mean ± SEM, *p < 0.05 by one-way ANOVA).