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: 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