Journal: JACC: Basic to Translational Science

Article Title: Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease

doi: 10.1016/j.jacbts.2025.101433

Figure Lengend Snippet: Nox4 Improves AVIC Survival During Calcification via Activation of elF2α/ATF4 Signaling (A) The effects of down-regulation of endogenous Nox4 with shNox4 adenovirus, or (B) overexpression of Nox4 with adNox4 adenovirus on protein levels of integrated stress response (ISR) markers elF2α phosphorylation and ATF4, ER chaperone KDEL, and cell survival markers CHOP and cleaved caspase-12 in cultured AVIC with or without OGM treatment. AVIC transfected with green fluorescent protein (GFP) virus or β-galactosidase (β-gal) virus as respective control cells. Mean data shown at the right. n = 3/group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with respective control cells (Con), # P < 0.05, ## P < 0.01, compared with OGM-stimulated AVIC transfected with GFP or β-gal virus. 2-way analysis of variance with a post hoc Tukey’s test. All data are mean ± SEM. CHOP = C/EBP homologous protein; elF2α = eukaryotic Initiation Factor 2; KDEL = peptide sequence Lys-Asp-Glu-Leu; other abbreviations as in .

Article Snippet: The primary antibodies used were: Nox4 (ab109225, Abcam); Runx2 (8486, Cell Signaling); Osteopontin (ab8448, Abcam); p-eIF2α (ab32157, Abcam); eIF2α (11170-1-AP, Proteintech); ATF4 (ab1371, Abcam); KDEL (ab69659, Abcam); Cleaved caspase-12 (2202, Cell Signaling); Caspase-12 (55238-1-AP, Proteintech); CHOP (ab11419, Abcam); GAPDH (10494-1-AP, Proteintech).

Techniques: Activation Assay, Over Expression, Phospho-proteomics, Cell Culture, Transfection, Virus, Control, Sequencing

Journal: JACC: Basic to Translational Science

Article Title: Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease

doi: 10.1016/j.jacbts.2025.101433

Figure Lengend Snippet: Guanabenz Attenuates AVIC Calcification Through Activation of elF2α/ATF4 Pathway (A) Calcium deposition by Alizarin Red staining and calcium concentration with or without Guanabenz (Gbz, 5 μmol/l, 14 days) treatment. Scale bar: 100 μm. Mean data shown at the right. (B) Immunoblots of calcification markers OPN and Runx2. (C) Western blots showing the effects of Gbz on expression of Nox4, p-eIF2α/ATF4 stress signaling, KDEL, and survival markers CHOP and cleaved caspase-12 in AVIC. Mean data shown at the right. n = 3-5/group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with respective control cells (Con), † P < 0.05, †† P < 0.01, compared with control cells without Gbz treatment, # P < 0.05, ## P < 0.01, compared with OGM without Gbz treatment. Two-way analysis of variance with a post hoc Tukey’s test. All data are mean ± SEM. Abbreviations as in , , .

Article Snippet: The primary antibodies used were: Nox4 (ab109225, Abcam); Runx2 (8486, Cell Signaling); Osteopontin (ab8448, Abcam); p-eIF2α (ab32157, Abcam); eIF2α (11170-1-AP, Proteintech); ATF4 (ab1371, Abcam); KDEL (ab69659, Abcam); Cleaved caspase-12 (2202, Cell Signaling); Caspase-12 (55238-1-AP, Proteintech); CHOP (ab11419, Abcam); GAPDH (10494-1-AP, Proteintech).

Techniques: Activation Assay, Staining, Concentration Assay, Western Blot, Expressing, Control

Journal: JACC: Basic to Translational Science

Article Title: Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease

doi: 10.1016/j.jacbts.2025.101433

Figure Lengend Snippet: Sephin1 Diminishes AVIC Calcification Through the Increase in elF2α Phosphorylation (A) Calcium deposition by Alizarin Red staining and calcium concentration with or without Sephin1 (0.5 μmol/L, 14 days) treatment. Scale bar: 100 μm. Mean data shown at the right. (B) Immunoblots of calcification markers OPN and Runx2 with Sephin1 treatment. (C) Western blots showing the activation of p-eIF2α/ATF4/KDEL stress signaling and Nox4 protein levels, and the decreases in apoptotic markers CHOP and cleaved caspase-12 in AVIC treated with Sephin1. Mean data shown at the right. n = 3-4/group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with respective control cells (Con), # P < 0.05, ## P < 0.01, ### P < 0.001, compared with OGM without Sephin1 treatment. Two-way analysis of variance with a post hoc Tukey’s test. All data are mean ± SEM. Abbreviations as in , , .

Article Snippet: The primary antibodies used were: Nox4 (ab109225, Abcam); Runx2 (8486, Cell Signaling); Osteopontin (ab8448, Abcam); p-eIF2α (ab32157, Abcam); eIF2α (11170-1-AP, Proteintech); ATF4 (ab1371, Abcam); KDEL (ab69659, Abcam); Cleaved caspase-12 (2202, Cell Signaling); Caspase-12 (55238-1-AP, Proteintech); CHOP (ab11419, Abcam); GAPDH (10494-1-AP, Proteintech).

Techniques: Phospho-proteomics, Staining, Concentration Assay, Western Blot, Activation Assay, Control

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.

Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

Techniques: Activation Assay, Western Blot, Staining, MANN-WHITNEY, Comparison, Binding Assay

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

Techniques: Transduction, Western Blot, Control, Binding Assay, In Vitro

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.

Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

Techniques: Activation Assay, Western Blot, Staining, MANN-WHITNEY, Comparison, Binding Assay

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

Techniques: Transduction, Western Blot, Control, Binding Assay, In Vitro