Journal: Journal of Advanced Research

Article Title: Impaired stemness in aging periodontal ligament stem cells is mediated by the progerin/endoplasmic reticulum stress/p53 axis

doi: 10.1016/j.jare.2024.10.029

Figure Lengend Snippet: Progerin accumulation in OPDLSCs resulted in ER stress activation and impairment of OPDLSC stemness. (a) The morphology of the endoplasmic reticulum in YPDLSCs transfected with the vector or the progerin plasmid was observed by transmission electron microscopy. (b) The UPR-target genes GRP78, PERK, IRE1α, ATF6, and CHOP expression in YPDLSCs transfected with the vector or the progerin plasmid. (c) The UPR-target proteins GRP78, PERK, p-PERK, IRE1, ATF6, and CHOP expression in YPDLSCs transfected with the vector or the progerin plasmid were detected by Western blotting. Statistical significance was defined as * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: PDLSCs were plated in a 6-well plate at a density of 2 × 10 5 , after the PDLSCs reached 80-90% confluence in the 6-well plate, they were serum-starved for 2 h. The OPDLSCs in the experimental group were transfected with 50 nM PERK (Santa Cruz Biotechnology, sc-36213, CA, USA), IRE1α (Santa Cruz Biotechnology, sc-40705), and ATF6 small interfering RNA (siRNA) (Genechem, Shanghai, China), and those in the control group were transfected with scrambled siRNA.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Transmission Assay, Electron Microscopy, Expressing, Western Blot

Journal: bioRxiv

Article Title: IRE1α is critical for kaempferol induced neuroblastoma differentiation

doi: 10.1101/432369

Figure Lengend Snippet: A. Gene expression analysis for ER stress marker genes with kaempferol pretreatment in IMR32 cells. The average ±SEM from three independent experiments. (*p < 0.05; ***p < 0.0o1; two-way ANOVA) B. Western blot analysis showing the expression of IRE1α and BiP expression on treatment with kaempferol 50 µM (KFL) and Brefeldin A 1 µg/ml (BFA) at 3, and 12 hour time points. C. Docked poses of ADP, kaempferol, and APY29 at the nucleotide binding site of human IRE1α kinase domain obtained using Autodock 4.0 and superimposed representation was made using PYMOL. D. LigPlot + analysis (2D representation) of the docked complexes of ADP, kaempferol and APY29. Red circles represent the common amino acid residues making interactions with all three compounds. Dashed green lines represent the hydrogen bonding between the amino acid residue and the compound. E. Docked poses of ADP, kaempferol and APY29 at DFG-in confirmation (D711, F712 and G713) of human IRE1α (PDB ID: 3P23). Superimposed docked poses and interaction analysis was performed using UCSF chimera. F. ADP Glo assay for kinase inhibition by kaempferol (KFL). Staurosporine (STS) was used as a positive control for kinase inhibition activity. Graphs representing the ±SEM of experiment performed in triplicate. G. Kinase inhibition assay performed using 10 µM and 100 µM of ATP with same range of concentrations of kaempferol. No significant change in the IC 50 value shows non-competitive mode of binding of kaempferol to the kinase domain of human IRE1α.

Article Snippet: The IRE1α (sc-40705-V) shRNA lentiviral particles was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knock down human IRE1α gene expression.

Techniques: Expressing, Marker, Western Blot, Binding Assay, Glo Assay, Inhibition, Positive Control, Activity Assay

Journal: bioRxiv

Article Title: IRE1α is critical for kaempferol induced neuroblastoma differentiation

doi: 10.1101/432369

Figure Lengend Snippet: A. Confirmation of knock down of IRE1α in IMR32 cells at mRNA level. The graph represents average ±SEM of experiment performed in triplicates. (*p < 0.05; one-way ANOVA) B. Western blot analysis showing IRE1α knockdown in IMR32 cells. IRE1α shRNA treated cells (KD) showing reduced expression than control shRNA (CON) treated cells. C. Immunocytochemistry for expression of β III tubulin showing reduced expression in IRE1α knockdown cells in the presence of kaempferol and APY29. Representative images of experiments performed more than three times. Scale bar = 67 µm. D. Western blot analysis for the expression of neuronal markers showing reduced expression in IRE1α knockdown cells. Reduction in the expression of XBP1s levels was also observed in IRE1α knockdown conditions. E. Phase contrast methylene blue stained images of IMR32 cells showing change in morphology and neurite outgrowth upon treatment with kaempferol (KFL) and APY29 for 96 h in control shRNA and IRE1α shRNA treated cells. Pretreatment with STF083010 (50 µM) inhibits the process of neuritogenesis further in IRE1α shRNA treated cells. Representative images of experiments performed more than three times. Scale bar = 100 µm. F. Graphs representing the number of cells with 2 or more neurite per cell in control shRNA and IRE1α shRNA treated IMR32 cells after 96 h incubation with kaempferol and APY29. STF083010 pretreatment reduces the number of neurite bearing cells in both kaempferol (KFL) and APY29 treated conditions further. The average ±SEM from 10 independent counting is shown.* represents comparison between control and kaempferol/APY29 treated cells; # and @ represents comparison between kaempferol/APY29 treated cells with STF083010 pretreated cells; $ and % represents comparison between kaempferol and APY29 treated conditions in control shRNA and IRE1α shRNA treated cells respectively (*p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA).

Article Snippet: The IRE1α (sc-40705-V) shRNA lentiviral particles was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) contain expression constructs encoding target-specific shRNA designed to specifically knock down human IRE1α gene expression.

Techniques: Western Blot, shRNA, Expressing, Immunocytochemistry, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: IRE1 is required in C2C12 differentiation. ( a ) IRE1-knockdown cell lines #1, #2, and mock cells were evaluated for the expression of IRE1/ Ern1 mRNA. Results are means + SEM (three biological replicates). ** p < 0.01. ( b ) Differentiation was induced in IRE1-knockdown cell lines and mock cells. After 48 h, cells were observed under phase contrast. Black arrows show the immature myotubes. Scale bar = 100 µm. ( c , d ) Cells were harvested after differentiation induction at 48 h. mRNA expression of myogenic factors ( c ) and UPR relative factors ( d ) was analyzed by qPCR. Results are means + SEM (three biological replicates). * p < 0.05, ** p < 0.01.

Article Snippet: IRE1α shRNA plasmid was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Knockdown, Expressing

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: IRE1 ribonuclease activity is required in early phase of C2C12 differentiation. ( a ) Differentiation was induced in the presence or absence of IRE1 RNase inhibitor, STF-083010 (60 µM; black bars) or DMSO (gray bars) for various time intervals as indicated. ( b ) Identification of critical time period for inhibitory effect of IRE1 activity on C2C12 differentiation. Scale bar = 200 µm. ( c ) Fusion index of STF-083010- or DMSO-treated cells. Results are mean + SEM (three biological replicates). The different letters denote significant differences between groups at p < 0.05 by Tukey’s HSD test.

Article Snippet: IRE1α shRNA plasmid was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: CDK5 is a downstream target of IRE1-XBP1 in C2C12 cells. ( a ) mRNA expression of Xbp1 and Cdk5 during C2C12 differentiation. Results are means + SEM (three biological replicates). Student’s t -test. ** p < 0.01. ( b ) An illustration of the predicted mouse Cdk5 promoter region. The region upstream of the Cdk5 gene comprises three XBP1-binding domains including CCAAT at −544 bp, TGCCACGTGG at −597 bp, and CCACGT at −1112 bp from the transcription start site. ( c , d ) Chromatin immunoprecipitation assay using a C2C12 genomic sample. Input DNA = positive control. Rabbit IgG was used as negative control ChIP ( c ). ChIP assay was performed by quantitative PCR analysis ( d ). Results are means + SEM (three biological replicates). Student’s t -test. * p < 0.05. ( e ) XBP1-knockdown and mock cells were transfected with the vector containing the Cdk5 promoter construct ( p 1400) or an empty vector. Cdk5 promoter activity was assessed by luciferase assay. Results are means + SEM (three biological replicates). Student’s t -test. ** p < 0.01.

Article Snippet: IRE1α shRNA plasmid was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Knockdown, Transfection, Plasmid Preparation, Construct, Activity Assay, Luciferase

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Expression levels of endogenous BI-1, IRE1α, XBP1 and CHOP post-HI. (A) Representative immunoblots showing protein expression levels in ipsilateral hemispheric tissue from 10-day-old neonatal rats. (B–E) Quantitative analysis of BI-1 (B), IRE1α (C), XBP1 (D) and CHOP (E) time-dependent expression after HI (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus 6 h, @ P <0.05 versus 24 h; n =4. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis.

Article Snippet: After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added.

Techniques: Expressing, Western Blot

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Ad-TMBIM6 administered at 48 h pre-HI reduced percentage infarcted area and showed localization to neurons and microglia at 72 h post-HI. (A) Representative images of TTC-stained sections and quantification of percentage infarcted area at 72 h post-HI in brain tissue from neonatal rats expressing BI-1 through adenoviral transduction with Ad-TMBIM6. Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle, n =6. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis. (B–E) Representative microphotographs of double immunofluorescence staining of BI-1 (red) with neurons (NeuN, green) (B), IRE1α (red) with neurons (NeuN, green) (C), BI-1 (red) with astrocytes (GFAP, green) (D) and BI-1 (red) with microglia (Iba-1, green) (E) in the peri-infarcted area at 72 h post-HI. Blue, DAPI. n =1/group. Merged images show localization of BI-1 or IRE1α to neurons, astrocytes or microglia. There was minimal localization of BI-1 to astrocytes. Scale bars: 50 μm.

Article Snippet: After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added.

Techniques: Staining, Expressing, Transduction, Double Immunofluorescence Staining

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Effects of BI-1 siRNA and IRE1α CRISPR activation plasmid on infarct area at 72 h post-HI. (A,B) Representative images of infarcted tissue (A) and quantification of percentage infarcted area (out of total brain area) (B) at 72 h post-HI. Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6; $ P <0.05 versus BI-1 siRNA; + P <0.05 versus Ad-TMBIM6+scramble siRNA; & P <0.05 versus IRE1α CRISPR AP; % P <0.05 versus control CRISPR. n =6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis. (C) Representative schematic of BI-1/IRE1α/XBP1/CHOP signaling pathway using the intervention groups (BI-1 siRNA and IRE1α CRISPR activation plasmid) to study the mechanism. (D) Representative immunofluorescence staining of BI-1 (red) with neurons (NeuN, green), astrocytes (GFAP, green) and microglia (Iba-1, green) after siRNA administration. Representative picture demonstrating brain region from where immunofluorescence pictures were obtained is shown upper right. n =1/group. Scale bars: 50 μm.

Article Snippet: After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added.

Techniques: CRISPR, Activation Assay, Plasmid Preparation, Immunofluorescence, Staining

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: BI-1 exerted its protective effects via the IRE1α–XBP1–CHOP pathway at 72 h post-HI. (A) Representative immunoblots of protein expression levels in ipsilateral hemispheric tissue from 10-day-old rats at 72 h post-HI. (B–F) Quantitative analysis of BI-1 (B), pIRE1α (C), XBP1 (D), CHOP (E) and ROMO1 (F) expression levels (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6; $ P <0.05 versus BI-1 siRNA; + P <0.05 versus Ad-TMBIM6+scramble siRNA; & P <0.05 versus IRE1α CRISPR AP; % P <0.05 versus control CRISPR; ^ P <0.05 versus Ad-TMBIM6+BI-1 siRNA; ε P <0.05 versus BI-1 siRNA-only group. n =6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis.

Article Snippet: After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added.

Techniques: Western Blot, Expressing, CRISPR

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Overexpression of BI-1 attenuated apoptosis at 72 h post-HI. (A) Representative immunoblots of protein expression levels in ipsilateral hemispheric tissue from 10-day-old rats at 72 h post-HI. (B–D) Quantification of Bcl-2 (B), BAX (C) and CC3 (D) expression level data (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6; $ P <0.05 versus BI-1 siRNA; + P <0.05 versus Ad-TMBIM6+scramble siRNA; & P <0.05 versus IRE1α CRISPR AP; % P <0.05 versus control CRISPR. n =6/group. One-way ANOVA followed by Tukey multiple-comparison, Holm–Sidak or Dunnett's post hoc analysis. (E) Representative microphotographs of positively CC3-stained (red) neurons (NeuN, green) from 10-day-old rats at 72 h post-HI. (F) Representative microphotographs of Fluoro-Jade C (FJC)-positive degenerating neurons in the peri-infarcted area. n =1/group. Scale bars: 100 µm.

Article Snippet: After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added.

Techniques: Over Expression, Western Blot, Expressing, CRISPR, Staining

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: BI-1 exerted its anti-apoptotic effects via inhibition of IRE1α in an in vitro OGD model. (A) Representative immunoblots of protein expression levels in PC12 cultured cells. Quantification of BI-1 (B), pIRE1α (C), pPERK (D) and CC3 (E) expression levels (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus control; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6 and Ad-TMBIM6+scramble siRNA; $ P <0.05 versus BI-1 siRNA; & P <0.05 versus APY-29; + P <0.05 DMSO; % P <0.05 versus CCT020312; ^ P <0.05 versus STF-083010 and control CRISPR. n =5–6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis.

Article Snippet: After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added.

Techniques: Inhibition, In Vitro, Western Blot, Expressing, Cell Culture, CRISPR

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: Down-regulation of IRE1α in the sciatic nerve after intrathecal injection of siRNA. ( A ) IRE1α was effectively reduced as measured by immunofluorescence. IRE1α was detected with an antibody against IRE1α, followed by a fluorescent-labeled secondary antibody (green). Data are expressed as mean ± SEM as the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( B ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against IRE1α. β-actin was used as loading control. Data are expressed as mean ± SEM indicated as the percentage of the respective controls and analyzed using Tamhane’s T2 analysis or unpaired Student’s t-test. Δ P < 0.05, ΔΔ P < 0.01, compared to control; ## P < 0.01, compared to DPN; * P < 0.05 (N = 6 per group).

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques: Injection, Immunofluorescence, Labeling, Western Blot, Control

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: Improved neurological morphology and function of sciatic nerve in DPN rats after the intrathecal injection of IRE1α siRNA. ( A ) Transfection of IRE1α siRNA increased the myelin sheath area and myelinated nerve fibers in DPN rats. Representative pictures of Luxol fast blue (LFB) staining (magnification 40×) of myelin sheath and ultrastructure (magnification 6000×) of myelinated nerve fibers. ( B ) Myelin sheath area expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( C ) The number of myelinated nerve fibers expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( D ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against PGP9.5. β-actin was probed as loading control. ( E ) The intensity of PGP9.5 expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( F ) NCV reported as mean ± SEM and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ΔΔ P < 0.01, compared to control; ## P < 0.01, compared to DPN; ** P < 0.01 (N = 6 per group).

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques: Injection, Transfection, Staining, Western Blot, Control

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: Inhibition of ER stress-related apoptosis in the sciatic nerve of DPN rats by intrathecal injection of IRE1α siRNA. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against P-IRE1α and XBP-1s. β-actin was probed as loading control. The intensity is expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( B ) TUNEL, p-JNK, and Caspae-12 were effectively eliminated as measured by immunohistochemistry method. Data are expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with Tamhane’s T2 analysis or unpaired Student’s t-test. ( C ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against GRP78, CHOP, Bcl-2, Bax and Cleaved-Caspase-3. β-actin was probed as loading control. Results of are expressed as mean ± SEM indicated as percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis (GRP78, Cleaved-Caspase-3, CHOP) or Tamhane’s T2 analysis (Bcl-2, Bax) or unpaired Student’s t-test. Δ P < 0.05, ΔΔ P < 0.01, compared to control; # P < 0.05, ## P < 0.01, compared to DPN; * P < 0.05, ** P < 0.01 (N = 6 per group).

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques: Inhibition, Injection, Western Blot, Control, TUNEL Assay, Immunohistochemistry

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: Expression of IRE1α, XBP-1s and GRP78 in RSC96 cells transfected with IRE1α siRNA and exposed to high glucose. ( A – C ) Representative Western blots using RSC96 cells and probed with antibodies against IRE1α, P- IRE1α, XBP-1s and GRP78. Results are expressed as mean ± SEM of the percentage of the respective 25 mM glucose group and analyzed using one-way ANOVA analysis. Δ P < 0.05, ΔΔ P < 0.01, compared to 25 mM glucose; # P < 0.05, ## P < 0.01, compared to 150 mM glucose; ★ P < 0.05, ★★ P < 0.01, Student’s unpaired t-test compared to 24 hours’ time point. * P < 0.05, ** P < 0.01 (n = 4 per group).

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques: Expressing, Transfection, Western Blot

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: High glucose-induced apoptosis and Ca 2+ levels in RSC96 cells after IRE1α siRNA transfection. ( A ) FACS apoptosis assay results using FITC-Annexin V and PI staining at 24 h and 48 h. ( B ) Intracellular Ca 2+ production measured with FACS analysis using Fluo-3 AM dye at 24 h and 48 h. ( C ) Apoptosis levels indicated as the percentage of the respective 25 mM glucose-treated cells. Data are expressed as mean ± SEM and analyzed using one-way ANOVA with LSD analysis. ( D ) Intracellular Ca 2+ levels indicated as the percentage of the respective 25 mM glucose-treated cells. Data are expressed as mean ± SEM and analyzed using one-way ANOVA with LSD analysis. Δ P < 0.05, ΔΔ P < 0.01, compared to25 mM glucose; ## P < 0.01, compared to 150 mM glucose; ★ P < 0.05, ★★ P < 0.01, Student’s unpaired t-test compared to 24 hours’ time point. * P < 0.05, ** P < 0.01 (n = 4 per group).

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques: Transfection, Apoptosis Assay, Staining

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: Expression of CHOP, Bcl-2, Bax, p-JNK, Caspase-12 and cleaved-Caspase-3 in RSC96 cells after IRE1α siRNA transfection and exposure to high glucose. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against CHOP, Bcl-2, Bax, p-JNK, Caspase-12 and cleaved-Caspase-3. ( B ) The band intensity expressed as mean ± SEM of the percentage of the respective 25 mM glucose-treated cells and analyzed using one-way ANOVA with LSD analysis (CHOP, Bcl-2/48 h, Bax/48 h, p-JNK/24 h, Caspase-12, cleaved-Caspase-3,) or Tamhane’s T2 analysis (p-JNK/48 h, Bcl-2/24 h, Bax/24 h, Caspase-3/24 h,). Δ P < 0.05, ΔΔ P < 0.01, compared to 25 mM glucose; # P < 0.05, ## P < 0.01, compared to 150 mM glucose; ★ P < 0.05, ★★ P < 0.01, Student’s unpaired t-test compared to 24 hours’ time point. * P < 0.05, ** P < 0.01 (n = 4 per group).

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques: Expressing, Transfection, Western Blot

Journal: Scientific Reports

Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

doi: 10.1038/s41598-018-20950-9

Figure Lengend Snippet: Schematic diagram depicting improvement of diabetic peripheral neuropathy due to relieved endoplasmic reticulum stress-induced apoptosis by IRE1α siRNA.

Article Snippet: IRE1α siRNA (sc-270028, Santa Cruz, USA) was prepared immediately prior to administration by mixing the siRNA solution with a transfection regent (sc-29528, Santa Cruz, USA), in a ratio of 1: 4 (v/v).

Techniques:

Journal: Viruses

Article Title: IRE1α Promotes Zika Virus Infection via XBP1

doi: 10.3390/v12030278

Figure Lengend Snippet: ZIKV infection activates IRE1α and induction of XBP1 targets. Cells were infected with ZIKV and RNA was harvested at the indicated number of days post infection (dpi). The relative mRNA abundance of ZIKV RNA ( A ) spliced XBP1 ( B ), ERDJ4 ( C ), and P58IPK ( D ) were determined by quantitative RT-PCR. Data are means ± SD of four replicates and are representative of at least two independent experiments. * p < 0.05, ** p < 0.001, by unpaired t test.

Article Snippet: IRE1α CRISPR/Cas9 knockdown cells were made by co-transfection of human IRE1α CRISPR/Cas9 KO plasmids and human IRE1α homology directed repair plasmids (Santa Cruz Biotechnology).

Techniques: Infection, Quantitative RT-PCR

Journal: Viruses

Article Title: IRE1α Promotes Zika Virus Infection via XBP1

doi: 10.3390/v12030278

Figure Lengend Snippet: IRE1α inhibitors prevent ZIKV-induced cell death. ( A + B ) Cells were treated with small molecule inhibitors or DMSO solvent control prior to infection with ZIKV. Viability was measured four days post-infection by quantifying ATP in metabolically active cells. ( A ) The IRE1α kinase inhibitor KIRA6 and nuclease inhibitor STF-083010 prevent loss of viability during ZIKV infection. ( B ) The IRE1α nuclease inhibitor 4μ8C, but not AMC, a structurally similar negative control compound, prevents ZIKV-induced loss of viability. ( C ) Wildtype (WT) and IRE1α knockodown (KD) cells were infected with ZIKV and viability was measured three days post-infection. Data are means ± SD of three replicates and are representative of at least two independent experiments. * p < 0.01, ** p < 0.001, by unpaired t test.

Article Snippet: IRE1α CRISPR/Cas9 knockdown cells were made by co-transfection of human IRE1α CRISPR/Cas9 KO plasmids and human IRE1α homology directed repair plasmids (Santa Cruz Biotechnology).

Techniques: Solvent, Control, Infection, Metabolic Labelling, Negative Control

Journal: Viruses

Article Title: IRE1α Promotes Zika Virus Infection via XBP1

doi: 10.3390/v12030278

Figure Lengend Snippet: IRE1α inhibitors reduce ZIKV replication. Cells were treated with small molecule inhibitors or DMSO solvent control prior to infection with ZIKV. RNA was harvested two days post-infection and the relative abundance of ZIKV RNA ( A + B ) and spliced XBP1 mRNA ( C + D ) were determined by quantitative RT-PCR. Data are means ± SD of three replicates and are representative of at least two independent experiments. The relative abundance of viral NS4B and vinculin loading control in cell lysates two days post-infection was determined by Western blotting and densitometry ( E ). The ratio of sXBP1 to vinculin is shown, normalized to uninfected cells ( F ). Data are means ± SD of three independent experiments. Viral titers in the cell culture medium were measured by plaque assay ( G ). PFU, plaque-forming units. Data are means ± SD of six replicates and are representative of three independent experiments. * p < 0.05, ** p < 0.01, by unpaired t test.

Article Snippet: IRE1α CRISPR/Cas9 knockdown cells were made by co-transfection of human IRE1α CRISPR/Cas9 KO plasmids and human IRE1α homology directed repair plasmids (Santa Cruz Biotechnology).

Techniques: Solvent, Control, Infection, Quantitative RT-PCR, Western Blot, Cell Culture, Plaque Assay

Journal: Viruses

Article Title: IRE1α Promotes Zika Virus Infection via XBP1

doi: 10.3390/v12030278

Figure Lengend Snippet: IRE1α and XBP1 promote ZIKV replication. ( A – D ) IRE1α or XBP1 knockdown (KD) cells generated using CRISPR-Cas9 were infected with ZIKV and RNA was harvested two days post-infection. The relative abundance of spliced XBP1 mRNA ( A ), ZIKV RNA ( B + D ), and ERDJ4 mRNA ( C ) were determined by quantitative RT-PCR. Data are means ± SD of three replicates and are representative of at least two independent experiments. * p < 0.05, ** p < 0.01, by unpaired t test.

Article Snippet: IRE1α CRISPR/Cas9 knockdown cells were made by co-transfection of human IRE1α CRISPR/Cas9 KO plasmids and human IRE1α homology directed repair plasmids (Santa Cruz Biotechnology).

Techniques: Knockdown, Generated, CRISPR, Infection, Quantitative RT-PCR

Journal: Viruses

Article Title: IRE1α Promotes Zika Virus Infection via XBP1

doi: 10.3390/v12030278

Figure Lengend Snippet: IRE1α inhibitors prevent ZIKV-induced ER reorganization. ( A + B ) Cells were infected with ZIKV for two days. Viral NS4B protein (red in merged image) and the ER marker, protein disulfide isomerase (PDI, green in merged image), were visualized by immunostaining. Nuclei were counterstained with TO-PRO-3 (blue). ( B ) Cells were treated with small molecule inhibitors or DMSO solvent control prior to infection with ZIKV and PDI staining, and ER reorganization was quantified. Data are means ± SD of three independent experiments. * p < 0.05, by unpaired t test.

Article Snippet: IRE1α CRISPR/Cas9 knockdown cells were made by co-transfection of human IRE1α CRISPR/Cas9 KO plasmids and human IRE1α homology directed repair plasmids (Santa Cruz Biotechnology).

Techniques: Infection, Marker, Immunostaining, Solvent, Control, Staining

Journal: Viruses

Article Title: IRE1α Promotes Zika Virus Infection via XBP1

doi: 10.3390/v12030278

Figure Lengend Snippet: Genetic disruption of IRE1α and XBP1 reduces ZIKV infection in a mouse model. Xbp1 flox/flox Ern1 flox/flox ESR Cre+ ( Xbp1 Δ Ire1α Δ) or Cre− littermate (WT) mice were treated with tamoxifen to induce the expression of Cre recombinase. Mice were given interferon receptor blocking MAR1-5A3 antibody the day before and after ZIKV infection. RNA was harvested from ( A ) kidney, ( B ) spleen, ( C ) testis, ( D ) eye, and ( E ) brain three days post-infection. ZIKV RNA was measured by quantitative RT-PCR and normalized to Hprt . Values represent mean ± SEM involving Cre- ( n = 11) and Cre+ ( n = 13) mice pooled from two independent experiments. Testes were obtained from the subset of mice that were male ( n = 9) for both Cre- and Cre+ animals. * p < 0.05 by Mann-Whitney test.

Article Snippet: IRE1α CRISPR/Cas9 knockdown cells were made by co-transfection of human IRE1α CRISPR/Cas9 KO plasmids and human IRE1α homology directed repair plasmids (Santa Cruz Biotechnology).

Techniques: Disruption, Infection, Expressing, Blocking Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: Molecular neurobiology

Article Title: Estrogen receptor β agonist attenuates endoplasmic reticulum stress-induced changes in social behavior and brain connectivity in mice

doi: 10.1007/s12035-018-0929-8

Figure Lengend Snippet: A) Treatment paradigm. Young adult male mice were injected intraperitoneally with tunicamycin (1mg/kg in dimethyl sulfoxide, DMSO) or vehicle (DMSO). Behavior tests were performed 12 h after tunicamycin injection. B) Tunicamycin treatment induced activation of IRE1. Phospho-IRE1 and IRE1 protein levels were determined in the mouse prefrontal cortex (PFC) 12 h after tunicamycin injection. Top. Representative blot. Bottom. Quantification of phospho-IRE1 to IRE1 ratio. Protein levels were measured by western blot analysis. **p < 0.01; Student's t-test. C) Tunicamycin treatment induced increase in spliced XBP1 (sXBP1) mRNA levels. mRNA levels of sXBP1 were determined by qRT-PCR in the mouse PFC 12 h after tunicamycin injection. The Cycle threshold (Ct) values were normalized to ribosomal protein S3 (RPS3). *p < 0.05; Student's t-test. D–E) Tunicamycin treatment induced deficits in social behavior. D) The three-chamber social interaction test. Left, time in chamber. ***p<0.001 vs. stranger mouse chamber. Two-way ANOVA (n=7 per group). Right, the discrimination index calculated as the difference in the time spent in the social and non-social chambers, divided by the sum of the time spent in both chambers. *p<0.05; Student's t-test (n=7 per group). E) Reciprocal social interaction test. *p<0.05; Student's t-test (n=7 per group). F) Schematic representation of stereotaxic injection of control or IRE1 shRNA lentiviral particles into mouse PFC followed by tunicamycin treatment for 12 h. G) Representative immunoblot data showing decrease in IRE1 expression in the PFC of mice injected with IRE1 shRNA particles in the presence or absence of tunicamycin. Tubulin was used as loading control. H–I) IRE1 shRNA administration attenuated tunicamycin-induced deficits in social behavior. H) The three-chamber social interaction test. Left, time in chamber. ***p<0.001 vs. stranger mouse chamber. Two-way ANOVA (n=6 per group). Right, the discrimination index calculated as the difference in the time spent in the social and non-social chambers, divided by the sum of the time spent in both chambers. *p<0.05 vs. con shRNA group; One-way ANOVA (n=6 per group). I) Reciprocal social interaction test. *p<0.05 vs. con shRNA group; One-way ANOVA (n=6 per group). Data are expressed as mean ±s.e.m. M, chamber housing stranger mouse; E, chamber housing an empty cage; C, center. ns, non-significant.

Article Snippet: IRE1 shRNA (m) Lentiviral (LV) particles and its control shRNA LV particles were purchased from Santa Cruz, CA, USA.

Techniques: Injection, Activation Assay, Western Blot, Quantitative RT-PCR, Control, shRNA, Expressing

Journal: Molecular neurobiology

Article Title: Estrogen receptor β agonist attenuates endoplasmic reticulum stress-induced changes in social behavior and brain connectivity in mice

doi: 10.1007/s12035-018-0929-8

Figure Lengend Snippet: A) Treatment paradigm. Young adult female mice were injected intraperitoneally with tunicamycin (1mg/kg in dimethyl sulfoxide, DMSO) or vehicle (DMSO) and behavioral testing was performed 12 hours after injection. B) Tunicamycin treatment induced decrease in phospho-IRE1 protein levels. Phospho-IRE1 and IRE1 protein levels were determined in the mouse PFC 12 h after tunicamycin injection. Top. Representative blot. Bottom. Quantification of phospho-IRE1 to IRE1 ratio. Protein levels were measured by western blot analysis. *p < 0.05; Student's t-test. C–D) No change in social behavior following tunicamycin treatment. C) The three-chamber social interaction test. Left, time in chamber. ***p<0.001 vs. stranger mouse chamber. Two-way ANOVA. Right, the discrimination index calculated as the difference in the time spent in the social and non-social chambers, divided by the sum of the time spent in both chambers. D) Reciprocal social interaction test. Data are expressed as mean ±s.e.m (n=6–8 per group). M, chamber housing stranger mouse; E, chamber housing an empty cage; C, center.

Article Snippet: IRE1 shRNA (m) Lentiviral (LV) particles and its control shRNA LV particles were purchased from Santa Cruz, CA, USA.

Techniques: Injection, Western Blot

Journal: Molecular neurobiology

Article Title: Estrogen receptor β agonist attenuates endoplasmic reticulum stress-induced changes in social behavior and brain connectivity in mice

doi: 10.1007/s12035-018-0929-8

Figure Lengend Snippet: A–B) Ovariectomy induces changes in social behavior. Adult sham–operated (SHAM), ovariectomized (OVX) mice and OVX-mice treated intraperitoneally with tunicamycin (1mg/kg) were tested for social behavior. A) The three-chamber social interaction test. Left, time in chamber. **p<0.01 vs. stranger mouse chamber. Two-way ANOVA (n=5 per group). Right, the discrimination index calculated as the difference in the time spent in the social and non-social chambers, divided by the sum of the time spent in both chambers. **p<0.01 vs. SHAM; One-way ANOVA (n=5 per group). B) Reciprocal social interaction test. **p<0.01 vs. SHAM; One-way ANOVA (n=5 per group). C–D) Adult male mice were injected intraperitoneally with tunicamycin (1mg/kg in DMSO) or vehicle (DMSO). Tunicamycin treatment induced decrease in (C) ERβ, but not (D) ERα protein levels in mouse PFC 12 h after injection. Top. Representative blot. Bottom. Quantification of ERβ protein. Protein levels were measured by western blot analysis and normalized to tubulin. ***p<0.001 vs. vehicle; Student's t test. E) No change in ERβ protein levels in PFC of female mice following tunicamycin injection. Top. Representative blot. Bottom. Quantification of ERβ or ERα protein. Protein levels were measured by western blot analysis and normalized to tubulin. F) Treatment paradigm. Adult male mice were injected intraperitoneally with tunicamycin (1mg/kg in dimethyl sulfoxide, DMSO), vehicle (DMSO), or ERB-041 (1mg/kg in DMSO, 30 minutes before tunicamycin injection) and tunicamycin (1mg/kg in DMSO). Behavior was performed 12 hours after tunicamycin injection. G) ERB-041 pretreatment attenuated tunicamycin-induced IRE1 activation. Phospho-IRE1 and IRE1 protein levels were determined in the mouse PFC 12 h after tunicamycin injection. Top. Representative blot. Bottom. Quantification of phospho-IRE1 to IRE1 ratio. Protein levels were measured by western blot analysis. **p<0.01 vs. vehicle, ##p<0.01 vs. tunicamycin group. One-way ANOVA. H) ERB-041 pretreatment attenuated tunicamycin-induced increase in CHOP (CCAAT/enhancer-binding protein homologous protein) mRNA levels. mRNA levels of CHOP were determined by qRT-PCR in the mouse prefrontal cortex (PFC) 12 h after tunicamycin injection. The Ct values were normalized to RPS3. **p<0.01 and ***p<0.001 vs. vehicle; ###p<0.001 vs. tunicamycin group. One-way ANOVA. Data are expressed as mean ±s.e.m. I–J) ERB-041 pretreatment attenuated tunicamycin-induced deficits in social behavior. I) The three-chamber social interaction test. Left, time in chamber. ***p<0.001 vs. stranger mouse chamber. Two-way ANOVA (n=8–10 per group). Right, the discrimination index calculated as the difference in the time spent in the social and non-social chambers, divided by the sum of the time spent in both chambers. *p<0.05 vs. vehicle, #p<0.01 vs. tunicamycin group; One-way ANOVA (n=8–10 per group). J) Reciprocal social interaction test. *p<0.05 vs. vehicle, #p<0.01 vs. tunicamycin group. One-way ANOVA (n=8–10 per group). Data are expressed as mean ±s.e.m. M, chamber housing stranger mouse; E, chamber housing an empty cage; C, center. ns, non-significant.

Article Snippet: IRE1 shRNA (m) Lentiviral (LV) particles and its control shRNA LV particles were purchased from Santa Cruz, CA, USA.

Techniques: Injection, Western Blot, Activation Assay, Binding Assay, Quantitative RT-PCR

Journal: Molecular neurobiology

Article Title: Estrogen receptor β agonist attenuates endoplasmic reticulum stress-induced changes in social behavior and brain connectivity in mice

doi: 10.1007/s12035-018-0929-8

Figure Lengend Snippet: Tunicamycin (1mg/kg in dimethyl sulfoxide, DMSO), vehicle (DMSO), or ERB-041 (1mg/kg in DMSO, 30 minutes before tunicamycin injection) and tunicamycin (1mg/kg in DMSO) was injected into mouse prefrontal cortex (PFC), and social behavior was examined at 12 h after tunicamycin administration. A) ERB-041 pretreatment attenuated tunicamycin-induced IRE1 activation. Phospho-IRE1 and IRE1 protein levels were determined in the mouse PFC 12 h after tunicamycin injection. Top. Representative blot. Bottom. Quantification of phospho-IRE1 to IRE1 ratio. Protein levels were measured by western blot analysis. *p < 0.05; On-way ANOVA. B–C) ERB-041 pretreatment attenuated tunicamycin-induced deficits in social behavior. B) The three-chamber social interaction test. Left, time in chamber. ***p<0.001 vs. stranger mouse chamber. Two-way ANOVA (n=7–8 per group). Right, the discrimination index calculated as the difference in the time spent in the social and non-social chambers, divided by the sum of the time spent in both chambers. *p<0.05 vs.vehicle; One-way ANOVA (n=7–8 per group). C) Reciprocal social interaction test. **p<0.01 vs. vehicle; One-way ANOVA (n=7–8 per group). Data are expressed as mean ±s.e.m. M, chamber housing stranger mouse; E, chamber housing an empty cage; C, center. ns, non-significant.

Article Snippet: IRE1 shRNA (m) Lentiviral (LV) particles and its control shRNA LV particles were purchased from Santa Cruz, CA, USA.

Techniques: Injection, Activation Assay, Western Blot

Journal: Molecular neurobiology

Article Title: Estrogen receptor β agonist attenuates endoplasmic reticulum stress-induced changes in social behavior and brain connectivity in mice

doi: 10.1007/s12035-018-0929-8

Figure Lengend Snippet: Tunicamycin treatment leads to the activation of IRE1 that promotes splicing of XBP1 mRNA. This results in increased levels of the transcription factor sXBP1, which triggers the expression of unfolded protein response (UPR) genes that induces alterations in brain connectivity and social behavior. The activation of ERβ signaling inhibits IRE1 activation, downregulates sXBP1 and attenuates ER stress-induced changes in brain connectivity and social behavior.

Article Snippet: IRE1 shRNA (m) Lentiviral (LV) particles and its control shRNA LV particles were purchased from Santa Cruz, CA, USA.

Techniques: Activation Assay, Expressing