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: Burns & Trauma

Article Title: Nuclear fragile X mental retardation-interacting protein 1-mediated ribophagy regulates immune function of dendritic cells in polymicrobial sepsis

doi: 10.1093/burnst/tkaf034

Figure Lengend Snippet: Septic exposure significantly upregulates UPR in dendritic cells, which is further enhanced by Nufip1 deficiency. ( a-b ) Splenic dendritic cells from WT and Nufip1 +/− mice were treated with either LPS or PBS for 24 h. (a) Western blotting was used to analyze the expression levels of HSPA5, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, ATF4, and DDIT3. (b) Representative confocal immunofluorescence images showing the ER morphology, scale bar: 10 μm. ( c-d ) Splenic dendritic cells were harvested from Nufip1 fl/fl and Cd11c cre Nufip1 fl/fl mice following CLP or sham surgery. (c) Western blotting analysis was performed to determine expression levels of HSPA5, p-EIF2A, EIF2A, ATF4, and DDIT3. (d) Representative confocal immunofluorescence images showing ER morphology, scale bar: 10 μm. Results are presented as mean ± standard deviation (SD). Statistical analysis was conducted using two-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. UPR unfolded protein response, WT wild-type, LPS lipopolysaccharide, PBS phosphate-buffered saline, HSPA5 shock protein family A member 5, EIF2AK3 eukaryotic translation initiation factor 2α kinase 3, EIF2A eukaryotic translation initiation factor 2A, ATF4 activating transcription factor 4, DDIT3 DNA damage-inducible transcript 3, ER endoplasmic reticulum, CLP cecal ligation and puncture

Article Snippet: Specific antibodies against the following proteins were used for western blotting (WB): NUFIP1 (Proteintech, Cat. No. 12515–1-AP), p62 (CST, #39749), LC3B (CST, #83506), HSPA5 (CST, #3177), phosphorylated EIF2AK3 (CST, #3179), EIF2AK3 (CST, #3192), ATF4 (CST, #11815), phosphorylated EIF2A (CST, #3398), EIF2A (CST, #5324), and DDIT3 (CST, #2895).

Techniques: Western Blot, Expressing, Immunofluorescence, Standard Deviation, Saline, Ligation

Journal: Burns & Trauma

Article Title: Nuclear fragile X mental retardation-interacting protein 1-mediated ribophagy regulates immune function of dendritic cells in polymicrobial sepsis

doi: 10.1093/burnst/tkaf034

Figure Lengend Snippet: NUFIP1-mediated ribophagy alleviates overactivated UPR in an EIF2AK3–ATF4–DDIT3 dependent manner. ( a-d ) Splenic dendritic cells from WT and Nufip1 +/− mice were pre-incubated with or without salubrinal, an EIF2A dephosphorylation inhibitor, for 30 min before LPS stimulation. (a) Flow cytometry was used to assess the expression of CD80, CD86, and MHC-II. (b) IL-12 p70 concentration in culture supernatant was determined using ELISA. (c) The percentage of dividing CD4 + T cells, as indicated by CFSE dye staining, was evaluated by flow cytometry. (d) Levels of IL-2, IL-4, and IFN-γ in co-culture supernatants were measured by ELISA. Data are presented as mean ± standard deviation (SD). Statistical significance was analyzed using two-way ANOVA followed by Tukey’s post hoc test. ns, not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001. NUFIP1 nuclear fragile X mental retardation-interacting protein 1, UPR unfolded protein response, EIF2AK3 eukaryotic translation initiation factor 2α kinase 3, ATF4 activating transcription factor 4, DDIT3 DNA damage-inducible transcript 3, WT wild-type, LPS lipopolysaccharide, IL interleukin, ELISA enzyme-linked immunosorbent assay, CFSE carboxyfluorescein succinimidyl ester, IFN interferon

Article Snippet: Specific antibodies against the following proteins were used for western blotting (WB): NUFIP1 (Proteintech, Cat. No. 12515–1-AP), p62 (CST, #39749), LC3B (CST, #83506), HSPA5 (CST, #3177), phosphorylated EIF2AK3 (CST, #3179), EIF2AK3 (CST, #3192), ATF4 (CST, #11815), phosphorylated EIF2A (CST, #3398), EIF2A (CST, #5324), and DDIT3 (CST, #2895).

Techniques: Incubation, De-Phosphorylation Assay, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Co-Culture Assay, Standard Deviation

Journal: Burns & Trauma

Article Title: Nuclear fragile X mental retardation-interacting protein 1-mediated ribophagy regulates immune function of dendritic cells in polymicrobial sepsis

doi: 10.1093/burnst/tkaf034

Figure Lengend Snippet: NUFIP1 modulates the EIF2AK3–ATF4–DDIT3 pathway by regulating ATF4 nuclear translocation. ( a ) Splenic dendritic cells were obtained from Nufip1 fl/fl and Cd11c cre Nufip1 fl/fl mice that underwent either CLP or sham surgery. Immunofluorescence imaging showing colocalization of NUFIP1 with the endoplasmic reticulum (ER-tracker), scale bar: 25 μm & 10 μm. ( b-d ) Splenic dendritic cells from WT and Nufip1 +/− mice were exposed to LPS or PBS for 24 h before additional analyses. (b) NUFIP1-binding proteins were extracted from total cell lysates by immunoprecipitation. (c) Immunoblotting of ATF4 levels in both the cytosol and nucleus, with β-actin and histone 3 serving as controls for cytosolic and nuclear proteins, respectively. (d) Laser confocal microscopy was used to evaluate ATF4 expression in dendritic cells, and the nuclei were stained using DAPI, scale bar: 10 μm. ( e ) A structure-based analysis of the protein interaction interface between NUFIP1 and ATF4. The images illustrate the predicted NUFIP1-ATF4 complex structure with labeled interaction hotspot residues. ( f ) Flag-binding proteins were extracted from total cell lysates by immunoprecipitation. Results are presented as mean ± standard deviation (SD). Statistical analysis was conducted using two-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01, **** p < 0.0001. NUFIP1 nuclear fragile X mental retardation-interacting protein 1, EIF2AK3 eukaryotic translation initiation factor 2α kinase 3, ATF4 activating transcription factor 4, DDIT3, DNA damage-inducible transcript 3, CLP cecal ligation and puncture, LPS, lipopolysaccharide, PBS phosphate-buffered saline, DAPI 4,6-diamidino-2-phenylindole, dihydrochloride

Article Snippet: Specific antibodies against the following proteins were used for western blotting (WB): NUFIP1 (Proteintech, Cat. No. 12515–1-AP), p62 (CST, #39749), LC3B (CST, #83506), HSPA5 (CST, #3177), phosphorylated EIF2AK3 (CST, #3179), EIF2AK3 (CST, #3192), ATF4 (CST, #11815), phosphorylated EIF2A (CST, #3398), EIF2A (CST, #5324), and DDIT3 (CST, #2895).

Techniques: Translocation Assay, Immunofluorescence, Imaging, Binding Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Expressing, Staining, Labeling, Standard Deviation, Ligation, Saline

Journal: Burns & Trauma

Article Title: Nuclear fragile X mental retardation-interacting protein 1-mediated ribophagy regulates immune function of dendritic cells in polymicrobial sepsis

doi: 10.1093/burnst/tkaf034

Figure Lengend Snippet: Dysregulated NUFIP1-mediated ribophagy impairs the immune function of dendritic cells in sepsis. NUFIP1-driven ribophagy alleviates the hyperactivated UPR via the EIF2AK3-ATF4-DDIT3 signaling pathway. Continuous septic exposure markedly impairs NUFIP1-mediated ribophagy, severely reducing dendritic cell function and resulting in notable immunosuppression following septic challenge. NUFIP1 nuclear fragile X mental retardation-interacting protein 1, UPR unfolded protein response, EIF2AK3 eukaryotic translation initiation factor 2α kinase 3, ATF4 activating transcription factor 4, DDIT3 DNA damage-inducible transcript 3, ER endoplasmic reticulum, HSPA5 shock protein family A member 5, LC3B microtubule-associated protein 1 light chain 3 β, ER stress endoplasmic reticulum stress

Article Snippet: Specific antibodies against the following proteins were used for western blotting (WB): NUFIP1 (Proteintech, Cat. No. 12515–1-AP), p62 (CST, #39749), LC3B (CST, #83506), HSPA5 (CST, #3177), phosphorylated EIF2AK3 (CST, #3179), EIF2AK3 (CST, #3192), ATF4 (CST, #11815), phosphorylated EIF2A (CST, #3398), EIF2A (CST, #5324), and DDIT3 (CST, #2895).

Techniques: Cell Function Assay

Journal: Experimental Animals

Article Title: Daphnetin ameliorates diabetic cardiomyopathy by regulating inflammation and endoplasmic reticulum stress-induced apoptosis

doi: 10.1538/expanim.24-0027

Figure Lengend Snippet: Daphnetin suppresses endoplasmic reticulum (ER) stress-induced apoptosis in diabetic rats. (A) The expression levels of GRP78, CHOP, p-PERK, and PERK were examined by western blotting. The ratio of p-PERK/PERK was calculated. GAPDH was utilized as the internal control. (B) GRP78 and CHOP expression levels were quantified by immunofluorescence staining. (C) The number of apoptotic cells in myocardial tissues was determined by TUNEL assay. (D) The levels of Bax, Bcl-2, and cleaved caspase-3 in myocardial tissues were examined by western blotting. GAPDH was utilized as the internal control. ## indicates P <0.01 compared to the control group. * indicates P <0.05 and ** indicates P <0.01 compared to the STZ group.

Article Snippet: After blocking, the membranes were probed with antibodies against collagen I (#ab270993; 1:1,000; Abcam, Cambridge, MA, USA), collagen III (#ab184993; 1:1,000; Abcam), B cell lymphoma-2 (Bcl-2; #26593-1-AP; 1:3,000; Proteintech), Bcl-2-associated X protein (Bax; #2772; 1:1,000; CST, USA), cleaved caspase-3 (#9661; 1:1,000; CST, Shanghai, China), GRP78 (#11587-1-AP; 1:2,000; Proteintech), CHOP (#15204-1-AP; 1:1,500; Proteintech), protein kinase R-like ER kinase (PERK; #3192; 1:1,000; CST), phosphorylated (p)-PERK (#3179; 1:1,000; CST), c-Jun N-terminal kinase (JNK; #17572-1-AP; 1:3,000; Proteintech), p-JNK (#4668; 1:1,000; CST), p38 mitogen-activated protein kinase (p38 MAPK; #9212; 1:1,000; CST), p-p38 MAPK (#28796-1-AP; 1:1,000; Proteintech).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, TUNEL Assay