Journal: Oncogene

Article Title: Endoplasmic Reticulum Stress Mediates Radiation-Induced Autophagy via PERK-eIF2α in Caspase-3/7 Deficient Cells

doi: 10.1038/onc.2010.74

Figure Lengend Snippet: (A) Wild-type (WT) and caspase-3/7 DKO MEF cells (caspase-3/7−/−) were transfected with 25nM siRNA against PERK, IRE1α and ATF6α. After 5hrs, cDNA GFP-LC3 (3µg) transfection was performed. The transfected cells were irradiated with 0 Gy or 5 Gy. After 48 hrs the percentage of cells with characteristic punctate GFP-LC3 fluorescence pattern was calculated relative to all GFP-positive cells. This was done in triplicate and error bar is shown as mean ± S.D. (B) Wild-type (WT) and caspase-3/7 DKO MEF cells were transfected with 25nM siRNA against PERK for 24hrs and were irradiated (0–6 Gy). After 8 days, surviving colonies were stained and scored. Values shown are the means ± S.D. of three separate repeated experiments. (C) PERK expression levels were determined by Western blotting using lysates from WT and caspase-3/7 DKO MEF cells treated with siRNA against control and PERK. Actin was probed to demonstrate equal loading. (D) IRE1α expression levels were determined by Western blotting using lysates from WT and caspase-3/7 DKO MEF cells treated with siRNA against control and IRE1α. Actin was probed to demonstrate equal loading. (E) ATF6α expression levels were determined by Western blotting using lysates from WT and caspase-3/7 DKO MEF cells treated with siRNA against control and ATF6α. Actin was probed to demonstrate equal loading.

Article Snippet: siRNA eIF2α, siRNA PERK, siRNA ATF6α, siRNA IRE1α (mouse), and siRNA control were purchased from Santa Cruz (SABT).

Techniques: Transfection, Irradiation, Fluorescence, Staining, Expressing, Western Blot, Control

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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naringenin ameliorates insulin resistance by modulating endoplasmic reticulum stress in hepatitis C virus-infected liver.

doi: 10.1016/j.biopha.2019.108848

Figure Lengend Snippet: Fig. 3. NGEN inhibits ER stress in HCVCP infected Huh-7.5.1 cell. (A–B) Huh-7.5.1 cells were infected with GFP or HCVCP adneovirus, then treated with or without 0.5 or 1 μM NGEN for 48 h, western blot assay was performed to detect the phosphorylation of IRE1α and the splicing of XBP1t, RT-qPCR was used to detect the mRNA levels of XBP1s and ERdj4. (C) Huh7.5.1 cells were infected with GFP or HCVCP adneovirus. 24 h after infection, cells were transfected with plasmid pERSE- luciferase reporter and treated with NGEN for another 24 h, the pRL-TK expressing Renilla luciferase was co-transfected as an internal control. The activity of ER stress pathway was monitored by a dual luciferase reporter assay system at 24 h after transfection. Huh7.5.1 cells were pre-treated with 0.5 or 1 μM NGEN for 24 h and then treated with 10 μg/mL tunicamycin for 6 or 12 h, (D) total cell lysates were immunoblotted with P-IRE1α, IRE1α, XBP1s and XBP1t, (E) the mRNA levels of XBP1s and ERdj4 were detected with RT-qPCR. The data are presented as the mean ± S.E. and expressed as fold-change relative to the level of cells transfected with control vector. *P < 0.05 vs. GFP or DMSO group, #P < 0.05 vs. HCVCP group.

Article Snippet: Human IRE1α shRNA expression vector (sc-40705-SH) and the negative control were purchased from Santa Cruz.

Techniques: Infection, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Expressing, Control, Activity Assay, Reporter Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Naringenin ameliorates insulin resistance by modulating endoplasmic reticulum stress in hepatitis C virus-infected liver.

doi: 10.1016/j.biopha.2019.108848

Figure Lengend Snippet: Fig. 4. NGEN ameliorates HCVCP induced IR by down-regulate the IRE1α levels. (A) Huh-7.5.1 cells were transfected with Flag, IRE1α, shCtrl and shIRE1α expression vectors, the IRE1α protein and mRNA levels were detected with western blot and RT-qPCR. (B) Huh-7.5.1 cells were transfected with expression vectors and treated with or without 1 μM NGEN for 48 h, after starved in serum free DMEM for 5 h, cells treated with or without 10 nM insulin for 5 min, western blot assay was performed to detect the phosphorylation of Akt. (D) The IRE1α protein and mRNA levels in HCVCP infected mice liver tissues were analyzed using western blot and RT-qPCR. The data are presented as the mean ± S.E. and expressed as fold-change relative to the level of cells transfected with control vector. *P < 0.05.

Article Snippet: Human IRE1α shRNA expression vector (sc-40705-SH) and the negative control were purchased from Santa Cruz.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Phospho-proteomics, Infection, Control, Plasmid Preparation

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

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: 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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

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: Expression of BI-1 improved long-term neurological deficits at 4 weeks post-HI. (A,B) In rat pups expressing BI-1 through treatment with Ad-TMBIM6 , long-term motor, learning and memory were assessed via foot fault test (A) and water maze test (B) at 4 weeks post-HI. (C,D) Quantitative analysis of percentage of weight of ipsilateral and contralateral regions out of total brain weights (C) and percentage of tissue loss (D) at 4 weeks post-HI. Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; n =6–9. One-way ANOVA followed by Tukey multiple-comparison or Holm–Sidak post hoc analysis.

Article Snippet: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

Techniques: Expressing, Hi-C

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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

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: Percentage cell viability significantly improved after transfection of PC-12 cells with Ad-TMBIM6 at MOI=100 in an in vitro OGD model. (A) Quantification of data shows time-dependent decrease in percentage cell viability for cultured PC12 cells when exposed to 1 h, 1.5, 3 h, 5 h and 6 h in OGD. (B) Quantification analysis of percentage cell viability of PC12 cells when exposed to varying concentrations (MOI) of viral vector. (C) Representative images of cell morphology and density of cells after OGD in all groups. Scale bar: 100 µm. (D) Quantification analysis of percentage cell viability with pathway interventions. Data presented as mean± s.d.; * P <0.05 versus control; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6+scramble siRNA (D) or versus MOI=100 (B); ε P <0.05 versus Ad-TMBIM6 only; $ P <0.05 versus BI-1 siRNA; & P <0.05 versus APY-29; + P <0.05 versus 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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

Techniques: Transfection, In Vitro, Cell Culture, Plasmid Preparation, 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: 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: BI-1 siRNA (sc-270613, Santa Cruz Biotechnology) and IREα CRISPR activation plasmid (AP) (sc-400576-ACT, Santa Cruz Biotechnology) were administered icv at 48 h pre-HI.

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