Journal: Cell stress & chaperones

Article Title: The Protective Role of the IRE1α/XBP1 Signaling Cascade in Autophagy During Ischemic Stress and Acute Kidney Injury.

doi: 10.1016/j.cstres.2025.02.004

Figure Lengend Snippet: Figure 2. Effects of IRE1α/XBP1 overexpression and inhibition on autophagy in HK-2 cells. (A)

Article Snippet: The following antibodies were utilized for western blotting: anti-β-actin (Wuhan Doctor De Biological Engineering Co., LTD, BM0627, 1:1000), anti-rabbit GRP78 (Abcam, ab108615, 1:500), anti-mouse P62 (Abcam, ab56416, 1:500), anti-rabbit LC3I/II (14/16 kDa) (Cell signaling technology, 12741T, 1:500), anti-rabbit IRE1α (Novus, NB100-2324, 1:300), anti-rabbit XBP1 (Abcam, ab37152, 1:500), anti-rabbit XBP1s (Cell signaling technology, 40435, 1:500), anti-rabbit Beclin1 (Abcam, ab210498, 1:500), and anti-rabbit Kim1 (is also known as Tim1) (Abcam, ab47635, 1:400).

Techniques: Over Expression, Inhibition

Journal: Cell stress & chaperones

Article Title: The Protective Role of the IRE1α/XBP1 Signaling Cascade in Autophagy During Ischemic Stress and Acute Kidney Injury.

doi: 10.1016/j.cstres.2025.02.004

Figure Lengend Snippet: Figure 3. Effects of XBP1 overexpression and IRE1α inhibition on GRP78 and autophagy-related

Article Snippet: The following antibodies were utilized for western blotting: anti-β-actin (Wuhan Doctor De Biological Engineering Co., LTD, BM0627, 1:1000), anti-rabbit GRP78 (Abcam, ab108615, 1:500), anti-mouse P62 (Abcam, ab56416, 1:500), anti-rabbit LC3I/II (14/16 kDa) (Cell signaling technology, 12741T, 1:500), anti-rabbit IRE1α (Novus, NB100-2324, 1:300), anti-rabbit XBP1 (Abcam, ab37152, 1:500), anti-rabbit XBP1s (Cell signaling technology, 40435, 1:500), anti-rabbit Beclin1 (Abcam, ab210498, 1:500), and anti-rabbit Kim1 (is also known as Tim1) (Abcam, ab47635, 1:400).

Techniques: Over Expression, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Potent and Selective Inhibitors of the Inositol-requiring Enzyme 1 Endoribonuclease

doi: 10.1074/jbc.m110.199737

Figure Lengend Snippet: FIGURE 1. Recombinant expression and activity of the purified cytosolic human IRE1 kinase endoribonuclease. A, expression strategy for a cytosolic fragment (amino acids 462–977) of human IRE1 (hIRE1-cyto) using a baculovirus/insect cell system. hIRE1-cyto was liberated from GST using PreScission protease. B, SDS-polyacrylamide gel electrophoresis analysis of GST-fused and PreScission-liberated hIRE1-cyto. C, schematic of the mini-XBP-1 stem-loop used as the hIRE1-cyto substrate for HTS and IC50 analysis; the Cy5 fluorophore was linked to the 5 end and black hole quencher 2 (BHQ2) was linked to the 3 end. D, time course analysis of mini-XBP-1 substrate cleavage (100 nM starting concentration) by the indicated concentrations of hIRE1-cyto.

Article Snippet: Proteins was transferred to a PVDF membrane and probed with rabbit anti-hIRE1 -cyto polyclonal antibody (18) or anti-hIRE1 phospho-specific (Ser724) antibody (Novus Biologicals).

Techniques: Recombinant, Expressing, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Potent and Selective Inhibitors of the Inositol-requiring Enzyme 1 Endoribonuclease

doi: 10.1074/jbc.m110.199737

Figure Lengend Snippet: FIGURE4.EnzymekineticsofhIRE1-cyto,mini-XBP-1RNAstem-loop,andtheinhibitorcompound3-ethoxy-5,6-dibromosalicylaldehyde.Titrationof the substrate Cy5-labeled mini-XBP-1 RNA stem-loop as shown in Fig. 1, with constant enzyme (hIRE1-cyto) concentration and measuring the slope of the initial reaction rates (A, i) determined the Michaelis-Menten constant (Km). Using a Lineweaver-Burk plot (A, ii), the x-axis intercept therefore specified the Km of 0.8 M for a single mini-XBP-1 RNA stem-loop substrate (Fig. 1C). Titration of both substrate and 3-ethoxy-5,6-dibromosalicylaldehyde demonstrated a non-competitive mode of inhibition relative to the substrate (B). Data in B, i, were plotted as 1/velocity against 1/substrate concentration as a Lineweaver-Burk plot (B, ii). The binding constants Kii and Kis were 71 and 88 nM, respectively. Data are shown from a representative of three separate experiments. Reaction kinetics data were fit using Visual Enzymics software.

Article Snippet: Proteins was transferred to a PVDF membrane and probed with rabbit anti-hIRE1 -cyto polyclonal antibody (18) or anti-hIRE1 phospho-specific (Ser724) antibody (Novus Biologicals).

Techniques: Labeling, Concentration Assay, Titration, Inhibition, Binding Assay, Software

Journal: Journal of Biological Chemistry

Article Title: Potent and Selective Inhibitors of the Inositol-requiring Enzyme 1 Endoribonuclease

doi: 10.1074/jbc.m110.199737

Figure Lengend Snippet: FIGURE 5. Surface plasmon resonance of hIRE1-cyto, substrate, and compound binding characteristics. Schematic of the XBP-1 RNA stem-loop with an extended stem used for surface plasmon resonance binding experiments is shown (A). Surface plasmon resonance binding profiles with the immobilized reagent on the solid chip surface are indicated at the bottom and the soluble binding partner indicated at the top (A–F). Active hIRE1-cyto was linked to the Biacore chip by amine coupling and demonstrated specific and dose-dependent binding to the substrate confirming that hIRE1-cyto was active on the chip (B). When compound 3-ethoxy-5,6-dibromosalicylaldehyde was passed over hIRE1-cyto on the chip, a specific dose-dependent binding was observed with fast on-fast off kinetics (C). The disassociation constant (Kd) was calculated to be 100 nM. Biotinylated stem-loop RNA was immobilized to a streptavidin- coated chip and a large mass change was observed when hIRE1-cyto was passed over (D). Repeated exposure to soluble hIRE1-cyto degraded the signal likely due to site-specific cleavage of the stem-loop RNA on the solid surface, therefore, only a single concentration is shown. Passage of 3-ethoxy-5,6- dibromosalicylaldehyde over a chip immobilized with the XBP-1 stem-loop RNA did not give rise to a detectable binding signal (E). When RNase A was coupled to the chip and compound 3-ethoxy-5,6-dibromosalicylaldehyde was passed over the surface, no detectable binding was observed (F).

Article Snippet: Proteins was transferred to a PVDF membrane and probed with rabbit anti-hIRE1 -cyto polyclonal antibody (18) or anti-hIRE1 phospho-specific (Ser724) antibody (Novus Biologicals).

Techniques: SPR Assay, Binding Assay, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Potent and Selective Inhibitors of the Inositol-requiring Enzyme 1 Endoribonuclease

doi: 10.1074/jbc.m110.199737

Figure Lengend Snippet: FIGURE 6. Inhibition of XBP-1 splicing in human cells using salicylaldehyde analogs. HEK293 cells were left untreated or treated with 300 nM Tg for 3 h. Compounds (right of panel) were added 2 h before Tg at the indicated dose; total RNA was harvested and RT-PCR was performed using human- specific XBP-1 primers flanking the splice site 3 h after stress induction. PCR products were run on 4% agarose gels and stained with ethidium bromide and shown as the inverse image. Spliced (S) and unspliced (U) reaction products of XBP-1 mRNA are designated (A). Human myeloma MM1.s cells were treated with 2 mM DTT as the ER stressing agent and exposed to increasing concentrations of the indicated compound for 2 h (B): both DTT and the compounds were added at the same time. When MM1.s cells were treated in 2% FCS medium as in B, the potency of 3-methoxy-6-bromosalicylaldehyde increased, indicating compounds are partially absorbed to serum proteins (C). Western blot showing relative levels of IRE1 in human cell lines both untreated and treated with 300 nM Tg for 2 h (D). MM1.s myeloma cells had high steady state levels of IRE1 compared with HEK293 and the IgE secreting human myeloma cell line U266. When HEK293 cells were incubated with Tg and increasing concentrations of 3-methoxy-6-bromosalicylaldehyde for 3 h, no change in phosphorylation status was observed (E). The dashed line is a reference to observe the slight decrease in mobility due to phosphorylation (p-hIRE1). The blot was reprobed with anti--tubulin antibody used as a loading control.

Article Snippet: Proteins was transferred to a PVDF membrane and probed with rabbit anti-hIRE1 -cyto polyclonal antibody (18) or anti-hIRE1 phospho-specific (Ser724) antibody (Novus Biologicals).

Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Staining, Western Blot, Incubation, Phospho-proteomics, Control

Journal: Cancer Medicine

Article Title: β‐asarone inhibits the migration, invasion, and EMT of bladder cancer through activating ER stress

doi: 10.1002/cam4.6059

Figure Lengend Snippet: β‐asarone (β‐as) s activates endoplasmic reticulum (ER) stress in BCa cells. (A) RNA‐seq data for 5637 cells treated with β‐as. BCa 5637 cells were treated with β‐as (240 μM), and total RNA was extracted for sequence analysis. (B) Analysis of gene ontology biological process (GOBP) database revealed enrichment of ER unfolded protein response. p < 0.001. (C) Expression of ER stress‐related protein markers (ATF6, PERK, IRE1, and BIP) in 253J and 5637 cells treated with different concentrations of β‐as (0, 120, 240, and 360 μM and 0, 240, 360, and 480 μM, respectively) for 24 h was analyzed by Western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001. (D) mRNA levels of ER stress‐related markers in 253J and 5637 cells treated with different concentrations (0, 120, 240, and 360 μM and 0, 240, 360, and 480 μM, respectively) of β‐as for 24 h were measured by q‐PCR. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Expression of BIP in 253J and 5637 cells that were mock‐treated or treated with β‐as (240 or 360 μM) was analyzed using immunofluorescence. Scale bar, 50 μm. *** p < 0.001. All data are presented as the mean ± SD of three independent assays.

Article Snippet: Primary rabbit antibodies against ATF6 (cat. no. ab37149; 1:2000), GRP78/BIP (cat. no. ab21685; 1:2000), and IRE1 (cat. no. ab124945; 1:2000), and secondary goat anti‐rabbit IgG (cat. no. ab205718; 1:2000) were purchased from Abcam.

Techniques: RNA Sequencing, Sequencing, Expressing, Western Blot, Immunofluorescence

Journal: Cancer Medicine

Article Title: β‐asarone inhibits the migration, invasion, and EMT of bladder cancer through activating ER stress

doi: 10.1002/cam4.6059

Figure Lengend Snippet: β‐asarone (β‐as) suppresses migration, invasion, and epithelial‐mesenchymal transition (EMT) of BCa cells via activating ER stress. (A) 253J and 5637 were treated without or with β‐as (240 or 360 μM) in the presence or absence of tauroursodeoxycholic acid (TUDCA; 10 μM) for 24 h and subjected to transwell migration and invasion assays, Magnification, ×100. Scale bar, 200 μm. *** p < 0.001. (B, C) Western blot analysis of the expression of ER stress‐related protein markers (ATF6, PERK, IRE1, and BIP) in 253J and 5637 cells treated as described in (A). * p < 0.05, ** p < 0.01, *** p < 0.001. (D, E) Western blot analysis of the protein expression of E‐cadherin, N‐cadherin, and vimentin in 253J and 5637 cells treated as described in (A). * p < 0.05, ** p < 0.01, *** p < 0.001. All data are presented as the mean ± SD of three independent assays.

Article Snippet: Primary rabbit antibodies against ATF6 (cat. no. ab37149; 1:2000), GRP78/BIP (cat. no. ab21685; 1:2000), and IRE1 (cat. no. ab124945; 1:2000), and secondary goat anti‐rabbit IgG (cat. no. ab205718; 1:2000) were purchased from Abcam.

Techniques: Migration, Western Blot, Expressing

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs activate UPR signaling to induce ERS . A HD11 cells were treated with 50–200 µg/mL OMVs for 6 h, and the relative mRNA levels of UPR downstream genes ATF4 , CHOP , EDEM1 , ERdj4 , and ATF6 were detected by qPCR. ( n = 3) B HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, and qPCR was used to detect the relative mRNA levels of UPR downstream genes ATF4 , CHOP , EDEM1 , ERdj4 , and ATF6 . ( n = 3) C , D HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, western blotting was used to detect the expression of PERK, p-PERK, eIF2α, p-eIF2α, CHOP, IRE1, p-IRE1, and ATF6 proteins in cell lysates, and gray value analysis was performed using ImageJ software ( D ). ( n = 3). E , F HD11 cells were treated with OMVs (100 µg/mL) for 6 h in the absence or presence of 4-PBA (2 mM), western blot analysis was used to detect the expression of PERK, p-PERK, eIF2α, p-eIF2α, CHOP, IRE1, p-IRE1, and ATF6 proteins in cell lysates, and gray value analysis was performed using ImageJ software ( F ). ( n = 3); n represents three biological replicates; data points indicate independent culture systems. Bar charts display mean ± standard error of the mean (SEM). Two-way ANOVA with Sidak correction was performed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Rabbit anti-p-IRE1 polyclonal antibody , Abmart, Shanghai, China , 1:5000.

Techniques: Western Blot, Expressing, Software

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: OMVs secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing ERS-mediated autophagy flux blockade . APEC-secreted OMVs, upon uptake by HD11 cells, induce ROS accumulation and Ca 2+ release, triggering ERS and activating UPR pathways, including the PERK, IRE1, and ATF6 signaling branches, leading to the expression of stress-related factors such as GRP78/BiP and CHOP. The sustained activation of ERS inhibits autophagosome degradation and disrupts the acidic environment of lysosomes, thereby preventing autophagosomes from fusing with lysosomes and impairing the phagocytic clearance capacity of macrophages. Collectively, these abnormal conditions facilitate APEC survival within HD11 cells and enable immune evasion, ultimately promoting bacterial dissemination and systemic infection in the host.

Article Snippet: Rabbit anti-p-IRE1 polyclonal antibody , Abmart, Shanghai, China , 1:5000.

Techniques: Infection, Expressing, Activation Assay