phosphorylated eif2α  (Cell Signaling Technology Inc)

Journal: Molecular Medicine Reports

Article Title: Naringin ameliorates intestinal injury in ulcerative colitis model mice by modulating the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2026.13805

Figure Lengend Snippet: Naringin inhibits JAK2/STAT3 signaling and restores tight junction proteins in dextran sulfate sodium-induced colitis. (A) Representative western blotting images of p-JAK2, JAK2, p-STAT3, STAT3, IL-6, occludin, ZO-1 and β-actin expression in colon tissues. Densitometric semi-quantification of the relative protein expression levels of (B) p-JAK2/β-actin, (C) JAK2/β-actin, (D) p-JAK2/JAK2, (E) p-STAT3/β-actin, (F) STAT3/β-actin, (G) p-STAT3/STAT3, (H) IL-6/β-actin, (I) occludin/β-actin and (J) ZO-1/β-actin. Data are presented as mean ± SEM. n=3. *P<0.05 and **P<0.01 vs. control group; # P<0.05 and ## P<0.01 vs. normal group. One-way ANOVA followed by Tukey's post-hoc test was applied. N, normal group; C, control group; Nar, naringin group; Mes, mesalazine group; p-, phosphorylated; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; IL-6, ZO-1, zona occludens-1.

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature, then incubated at 4°C overnight with primary antibodies against IL-6 (1:1,000; cat. no. Bs-0782R; BIOSS), phosphorylated (p-)JAK2 (1:1,000; cat. no. bs-2485R; BIOSS), JAK2 (1:1,000; cat. no. bs-0908R; BIOSS), p-STAT3 (1:1,000; cat. no. bs-1658R; BIOSS), STAT3 (1:1,000; cat. no. bs-55208R; BIOSS), occludin (1:1,000; cat. no. A2601; ABclonal Biotech Co., Ltd.), zona occludens-1 (ZO-1; 1:1,000; cat. no. A0659; ABclonal Biotech Co., Ltd.) and β-actin (1:1,000; cat. no. ab8227; Abcam).

Techniques: Western Blot, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: Naringin ameliorates intestinal injury in ulcerative colitis model mice by modulating the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2026.13805

Figure Lengend Snippet: Naringin suppresses JAK2/STAT3 activation in IL-6-stimulated Caco-2 cells with STAT3 silencing. (A) Western blot analysis of p-JAK2, JAK2, p-STAT3, STAT3, occludin and ZO-1 in Caco-2 cells under indicated treatments. (B) Validation of STAT3 knockdown efficiency: Relative STAT3 expression in cells transfected with siSTAT3 vs. siNC. ## P<0.01 vs. siNC. Densitometric semi-quantification of relative protein expression levels of (C) p-JAK2/β-actin, (D) JAK2/β-actin, (E) p-JAK2/JAK2, (F) p-STAT3/β-actin, (G) STAT3/β-actin, (H) p-STAT3/ STAT3, (I) occludin/β-actin, (J) ZO-1/β-actin. Data are presented as mean ± SEM. n=3. *P<0.05 and **P<0.01 vs. IL-6 group; ## P<0.01 vs. normal group; Δ P<0.05 and ΔΔ P<0.01 IL-6 + Nar group vs. IL-6 + Nar + siSTAT3 group. One-way ANOVA followed by Tukey's post-hoc test was applied. N, normal group; Nar, naringin group; Mes, mesalazine group; p-, phosphorylated; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; ZO-1, zona occludens-1; si, small interfering RNA; NC, negative control.

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature, then incubated at 4°C overnight with primary antibodies against IL-6 (1:1,000; cat. no. Bs-0782R; BIOSS), phosphorylated (p-)JAK2 (1:1,000; cat. no. bs-2485R; BIOSS), JAK2 (1:1,000; cat. no. bs-0908R; BIOSS), p-STAT3 (1:1,000; cat. no. bs-1658R; BIOSS), STAT3 (1:1,000; cat. no. bs-55208R; BIOSS), occludin (1:1,000; cat. no. A2601; ABclonal Biotech Co., Ltd.), zona occludens-1 (ZO-1; 1:1,000; cat. no. A0659; ABclonal Biotech Co., Ltd.) and β-actin (1:1,000; cat. no. ab8227; Abcam).

Techniques: Activation Assay, Western Blot, Biomarker Discovery, Knockdown, Expressing, Transfection, Small Interfering RNA, Negative Control

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: FCN3 regulated Wnt/β-catenin signaling to influence Treg activation. ( A - B ) qRT-PCR and WB analysis of APC and β-catenin in HCC tumor and adjacent non-tumor tissues. ( C ) Clinical sample IF staining showing β-catenin expression and localization with DAPI nuclear counterstain. ( D - E ) The levels of APC and β-catenin in HCC cell lines were assessed by qRT-PCR and WB. ( F - G ) qRT-PCR and WB evaluation of APC and β-catenin in Hep3B cells treated with OE-NC, OE-FCN3, OE-FCN3 + DMSO, or OE-FCN3 + LY2090314 (β-catenin activator). ( H ) The levels of TGF-β1 and IL-10 in cell culture supernatant of each group were examined by ELISA. ( I - M ) Wound healing, Transwell and flow cytometry assays evaluating the effect of OE-FCN3 and OE-FCN3 + LY2090314 on cell migration, invasion and apoptosis of Hep3B cells. ( N ) Flow cytometry was performed to analyze the effects of OE-FCN3 and OE-FCN3 + LY2090314 treatment in Hep3B cells on the proportion of CD4⁺CD25⁺FOXP3 + Treg cells in Hep3B-PBMC co-cultures. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. N/ THLE-2/ OE-NC/ OE-FCN3 + DMSO

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against FCN3 (#ABIN521997, 1:2000; Antibodies Online, Aachen, Germany), FOXP3 (#22228-1-AP, 1:500; Proteintech), CD25 (#83896-1-RR, 1:1000; Proteintech), APC (#sc-9998, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (#66379-1-Ig, 1:5000; Proteintech), phosphorylated β-catenin (p-β-catenin) (#80067-1-RR, 1:1000; Proteintech) or STT3A (#ab320831, 1:20000; Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: N-glycosylated FCN3 influenced Wnt/β-catenin signaling and Treg activation. ( A ) qRT-PCR analysis of APC and β-catenin mRNA levels in Hep3B cells transfected with FCN3-WT or FCN3-N189Q. ( B ) WB analysis showing the levels of APC, β-catenin, and p-β-catenin. ( C ) Subcellular localization of β-catenin was analyzed by WB. ( D ) ELISA was conducted to assess the levels of TGF-β1 and IL-10 in cell culture supernatant of Hep3B cells transfected with FCN3-WT or FCN3-N189Q. ( E - K ) The effects of FCN3 glycosylation on cell viability, migration, invasion and apoptosis in Hep3B cells were respectively evaluated by CCK-8, wound healing, Transwell and flow cytometry assays. ( L ) Flow cytometry demonstrating the impact of FCN3 glycosylation on the proportion of CD4⁺CD25⁺FOXP3 + Treg cells in Hep3B-PBMC co-cultures. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. FCN3-WT

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against FCN3 (#ABIN521997, 1:2000; Antibodies Online, Aachen, Germany), FOXP3 (#22228-1-AP, 1:500; Proteintech), CD25 (#83896-1-RR, 1:1000; Proteintech), APC (#sc-9998, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (#66379-1-Ig, 1:5000; Proteintech), phosphorylated β-catenin (p-β-catenin) (#80067-1-RR, 1:1000; Proteintech) or STT3A (#ab320831, 1:20000; Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Glycoproteomics, Migration, CCK-8 Assay, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

doi: 10.1007/s13402-025-01159-1

Figure Lengend Snippet: STT3A involved in HCC progression via promoting FCN3 N-glycosylation. ( A ) Bioinformatics analysis revealing STT3A expression in normal and HCC liver tissues. ( B - D ) qRT-PCR, WB and IHC assays were performed to assess STT3A levels in HCC tumor and adjacent non-tumor tissues. ( E - F ) The mRNA and protein levels of STT3A in normal hepatocyte and HCC cell lines were analyzed by qRT-PCR and WB. ( G ) Correlation analysis between STT3A protein expression and FCN3 protein expression in tumor tissues from HCC patients. ( H ) Co-IP assay verified STT3A-FCN3 interaction. ( I ) WB analysis of FCN3 glycosylation in Hep3B cells transfected with sh-STT3A or sh-NC. ( J - K ) The effects of sh-STT3A on the expression of APC and β-catenin in Hep3B cells were examined by qRT-PCR and WB. ( L ) ELISA showing TGF-β1 and IL-10 levels in Hep3B cell supernatants following sh-STT3A or sh-NC treatment. ( M - Q ) Wound healing, Transwell and flow cytometry assays were conducted to evaluate the effects of sh-STT3A on Hep3B cell migration, invasion and apoptosis. ( R ) The influence of sh-STT3A on the proportion of CD4⁺CD25⁺FOXP3 + Treg cells in Hep3B-PBMC co-cultures was assessed by flow cytometry. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. N/ THLE-2/ sh-NC

Article Snippet: After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against FCN3 (#ABIN521997, 1:2000; Antibodies Online, Aachen, Germany), FOXP3 (#22228-1-AP, 1:500; Proteintech), CD25 (#83896-1-RR, 1:1000; Proteintech), APC (#sc-9998, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (#66379-1-Ig, 1:5000; Proteintech), phosphorylated β-catenin (p-β-catenin) (#80067-1-RR, 1:1000; Proteintech) or STT3A (#ab320831, 1:20000; Abcam, Cambridge, MA, USA).

Techniques: Glycoproteomics, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Migration

Journal: International Journal of Oncology

Article Title: Ephrin-B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/β-catenin signaling pathway

doi: 10.3892/ijo.2025.5821

Figure Lengend Snippet: Wnt/β-catenin signaling pathway is responsible for EFNB2-mediated biological effects in gastric cancer cells. (A) Gene Expression Profiling Interactive Analysis and (B) Tumor Immune Estimation Resource databases were employed to explore the transcriptional correlation between EFNB2 and CTNNB1, GSK3β and downstream MYC (Spearman). Representative western blot bands and semi-quantification of protein expression levels of p-GSK3β, GSK3β, β-catenin and c-myc detected under the condition of EFNB2 (C) knockdown and (D) overexpression (one-way ANOVA for AGS cells; unpaired Student's test for HGC-27 cells). Representative western blot bands and semi-quantification of protein expression levels in the presence of (E) an agonist (CHIR99021) and (F) an inhibitor (DIF-3) of the Wnt/β-catenin signaling pathway in the EFNB2 knockdown or overexpression groups, respectively. Protein levels of p-GSK3β, GSK3β, β-catenin and c-myc were detected by western blotting (one-way ANOVA). Data are presented as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. sh-NC or vector group. ns, not significant; NC, negative control; DIF-3, differentiation-inducing factor-3; p-, phosphorylated; OE, overexpression vector; sh, short hairpin RNA; CTNNB1, catenin β1; TPM, transcript per million; EFNB2, ephrin-B2.

Article Snippet: The details of the primary antibodies used were as follows: EFNB2 (1:1,000; cat. no. sc-398735; Santa Cruz Biotechnology, Inc.), Bcl-2 (1:2,000; cat. no. ab182858; Abcam), Bax (1:2,000; cat. no. ab32503; Abcam), CyclinD1 (1:1,000, cat. no. 2978; Cell Signaling Technology, Inc.), CDK4 (1:1,000; cat. no. 12790; Cell Signaling Technology, Inc.), GSK3β (1:5,000; cat. no. 82061-1-RR; Proteintech Group, Inc.), phosphorylated (p-)GSK3β (Ser389; 1:1,000; cat. no. 14850-1-AP; Proteintech Group, Inc.), β-catenin (1:5,000; cat. no. 51067-2-AP; Proteintech Group, Inc.), c-myc (1:5,000; cat. no. 808451-RR; Proteintech Group, Inc.) and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.).

Techniques: Gene Expression, Western Blot, Expressing, Knockdown, Over Expression, Plasmid Preparation, Negative Control, shRNA

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: Genes & Diseases

Article Title: Targeting fatty acid synthase to overcome PARP inhibitor resistance and to create an artificial synthetic lethality for triple-negative breast cancer

doi: 10.1016/j.gendis.2025.101817

Figure Lengend Snippet: Combination synergy on DNA damage, NHEJ repair, and apoptosis. (A, B) Western blotting and quantification analyses of FASN, PARP1, γ-H2AX, and actin loading control in MDA-MB-231 and MDA-MB-436 cells treated with 5HLS, talazoparib, or the combination. (C, D) The fraction of maximum γ-H2AX induction derived using the Bliss-compatible scaling formula. (E) Host cell reactivation assay of NHEJ activity in MDA-MB-231 and MDA-MB-436 cells treated with 5HLS, talazoparib, or the combination. (F) Comparison between the observed and expected NHEJ activity inhibition by the combination using the Bliss independence model. (G) Caspasae3/7 activity assay of MDA-MB-231 and MDA-MB-436 cells treated with 5HLS, talazoparib, or the combination. (H) The fraction of maximum caspase 3/7 activation derived using the Bliss-compatible scaling formula. (I) Annexin V staining as an indicator of apoptosis in MDA-MB-231 and MDA-MB-436 cells following treatments with 5HLS, talazoparib, or the combination. (J) The fraction of maximum apoptosis induction derived using the Bliss-compatible scaling formula. n = 3; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Antibodies against PARP1 (#66520) and phosphorylated histone H2AX (γH2AX) (#613402) were from Proteintech (Rosemont, Illinois, USA) and BioLegend (San Diego, California, USA), respectively.

Techniques: Western Blot, Control, Derivative Assay, Host-Cell Reactivation, Activity Assay, Comparison, Inhibition, Activation Assay, Staining

Journal: Genes & Diseases

Article Title: Targeting fatty acid synthase to overcome PARP inhibitor resistance and to create an artificial synthetic lethality for triple-negative breast cancer

doi: 10.1016/j.gendis.2025.101817

Figure Lengend Snippet: In - vivo effect of 5HLS and talazoparib combination on tumor growth. (A) Tumor volume and body weight of mice treated by vehicle (Veh), 5HLS, talazoparib (Tal), or combination (Comb) of 5HLS and talazoparib. (B, C) Gross anatomy (B) and wet weight (C) of dissected xenograft tumors at the end of the study. (D) Synergy analysis. Tumor growth inhibition (TGI) was derived using the wet weight of tumors. The expected combination inhibition (C (E) ) was calculated from that of 5HLS and talazoparib alone using the Bliss independence model (see Materials and Methods ). C (O) represents the observed combination inhibition. (E) Immunohistochemical analyses of FASN, PARP, and γH2AX. Scale bar, 50 μm. (F) Western blotting analyses of FASN, BRCA1, PARP1, cleaved PARP1 (cPARP1), γH2AX, and actin loading control in xenograft tumors from mice treated with vehicle, 5HLS, talazoparib, and the combination of 5HLS and talazoparib. Each lane represents mixed samples of five tumors in equal proportion within the treatment group. n = 5; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Antibodies against PARP1 (#66520) and phosphorylated histone H2AX (γH2AX) (#613402) were from Proteintech (Rosemont, Illinois, USA) and BioLegend (San Diego, California, USA), respectively.

Techniques: In Vivo, Inhibition, Derivative Assay, Immunohistochemical staining, Western Blot, 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: 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