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  • 95
    Thermo Fisher gene exp xbp1 hs00231936 m1
    The 4‐phenylbutyrate (4‐PBA) reduced tunicamaycin‐induced resistin and ER stress markers messenger ribonucleic acid (mRNA) in THP‐1 human monocytes. THP‐1 cells were pre‐incubated with 750, 1,500, 3,000 or 7,500 μmol/L of 4‐PBA, a chemical chaperone, for 14 h, and 0.1 μg/mL tunicamycin was subsequently added to the medium for 24 h. The mRNA of (a) endoplasmic reticulum stress markers (immunoglobulin heavy chain‐binding protein [BiP], pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase [PERK], activating transcription factor 4 [ATF4], CAAT/enhancer binding protein‐α homologous protein [CHOP], ATF6, inositol requiring enzyme 1 [IRE1] and X‐box binding protein 1 <t>[XBP1])</t> and (b) resistin ( RETN ) were quantitated using reverse transcription polymerase chain reaction and shown relative to 18S rRNA mRNA as described in the Materials and Methods. The mRNA of control (4‐PBA [–], tunicamycin [–]) was defined as 1.0. The mean ± standard error of the mean of five separate experiments with triplicate samples is shown. anova ; P = 8.49 × 10 −8 (BiP), P = 0.00040 (PERK), P = 2.46 × 10 −6 (ATF4), P = 1.53 × 10 −5 (CHOP), P = 0.00085 (ATF6), P = 0.0070 (IRE1), P = 0.00016 <t>(XBP1),</t> P = 5.44 × 10 −6 (RETN); Scheffe's test; †† P
    Gene Exp Xbp1 Hs00231936 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology xbp1
    Integration of ChIPmentation and RNA-seq data reveals <t>XBP1</t> direct target genes and its regulatory network. a Venn diagram comparing previously reported XBP1 target genes of other secretory cell types with the Th2 direct target genes of this study. XBP1 direct target genes of this study are those that are common in both “XBP1-occupied genes in Th2” and “Differentially expressed genes (Th2 → Th2+4μ8c)” categories. The XBP1 direct target genes of B cell/plasma cell, skeletal muscle cells, and pancreatic β-cells were as observed by Acosta-Alvear et al. [ 17 ] and have been used here for comparison. b Heatmap showing the pattern of XBP1 direct target gene expression. The top 38 genes that follow a distinct pattern have been displayed. c Transcriptional regulatory network: transcription factors that are direct target of XBP1. The genes in the network are differentially expressed (upregulated—red; downregulated—blue) up on 4μ8c treatment. The transcription factors that are not differentially expressed but have a XBP1 ChIPseq peak are shown in the right-hand side list
    Xbp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 853 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    xbp1  (Abcam)
    92
    Abcam xbp1
    ZIKV infection induces the splicing of <t>xbp1</t> and the translocation of XBP1s into the nuclei of human neural cells. a , b CCF- STTG1 and SK-N-SH were infected with ZIKV at an MOI of 5 PFU/cell. Positive control (Tm) was treated with 2 μM tunicamycin. RNA was extracted at 24 and 48 hpi and the first strand cDNA was synthesized. Primers that specifically amplified xbp1s and total xbp1 (including both xbp1u and xbp1s ) were used to analyze xbp1s via RT-qPCR. The relative expression levels of the xbp1s and t-xbp1 were calculated according to the 2 −∆∆ Ct method. The ratios of xbp1s / t-xbp1 between mock-infected and ZIKV-infected were calculated. Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P
    Xbp1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti xbp1
    Hcy induces ER stress and inflammation and upregulates Ero1α in the thoracic aortas of HHcy mice. (A) The lysates of the thoracic aortas in normal (Ctrl) and HHcy mice were subjected to immunoblotting analysis for spliced <t>XBP1</t> (XBP1s), unspliced XBP1 (XBP1u), phosphorylated eIF2α (p-eIF2α), and eIF2α. Representative blots were shown. Statistical analysis for XBP1s/XBP1u and p-eIF2α/eIF2α was indicated. Data were presented as mean ± SEM from seven biological replicates, **p
    Anti Xbp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend purified anti xbp 1s
    Glutamate stimulation induces local activation of the IRE 1‐ XBP 1 pathway in distal dendrites. Primary cultured mouse hippocampal neurons maintained for 10 days were used for the analyses. (a) Immunofluorescence staining analysis of IRE 1 (green) and MAP 2 (dendritic marker: red). IRE 1 was localized to MAP 2‐positive dendrites (Bar: 20 μm). (b) Western blot analysis of phosphorylated IRE 1 (P‐ IRE 1), IRE 1, phosphorylated eIF 2α (P‐ eIF 2α) and eIF 2α. Cells were treated with 7.5 μM glutamate for the indicated times. P‐ IRE 1 and P‐ eIF 2α were transiently up‐regulated following glutamate stimulation. (c) RT ‐ PCR analysis of Xbp1 and Bip . Spliced Xbp1 ( <t>Xbp1s</t> ) and Bip were transiently induced by treatment with 7.5 μM glutamate for the indicated times ( Xbp1u : unspliced Xbp1 ). (d) Immunofluorescence staining analysis of IRE 1 (green), MAP 2 (blue) and PSD 95 (postsynaptic marker: red). IRE 1 was observed at MAP 2‐positive neurites and PSD 95‐positive postsynaptic sites of dendrites. The localization and level were not significantly changed by the treatment with 7.5 μM glutamate for 1.5 h. The lower panels show high magnification of the dendrites shown in the upper panels. (Bar in upper panels: 20 μm, in lower panels: 5 μm). (e) Immunofluorescence staining analysis of P‐ IRE 1 (green), MAP 2 (blue) and PSD 95 (red). IRE 1 was phosphorylated at PSD 95‐positive postsynaptic sites of dendrites in response to 7.5 μM glutamate exposure for 1.5 h. The lower panels show high magnification of the dendrites shown in the upper panels. Arrow heads indicate immunoreactivities of P‐ IRE 1 overlapped with those of PSD 95. (Bar in upper panels: 20 μm, in lower panels: 5 μm). (f) P‐ IRE 1 fluorescence intensities 40–90 μm from the somata of the dendrites in (e). The fluorescence intensities in these dendrites were not increased by glutamate treatment (mean ± SD , N = 10; number of cells prepared from five independent cultures). (g) The number of P‐ IRE 1‐positive puncta overlapping with PSD 95‐positive postsynaptic sites 40–90 μm from the somata in (e). The number of P‐ IRE 1‐positive postsynaptic sites was increased by glutamate exposure (mean ± SD , N = 10; number of cells prepared from five independent cultures; *** p
    Purified Anti Xbp 1s, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc xbp1
    Induction of the ER-UPR complex in cells expressing mutant TULP1. (A) Western blot analysis using antibodies against BiP, phosphorylated PERK (pPERK), <t>XBP1</t> and XBP1s, showed induction of UPR stress proteins in cells expressing mutant forms of TULP1. Actin was used a protein loading control. Western blot experiments were repeated twice. (B) Quantification of ER-stress protein markers was performed using densitometry in mutant TULP1 expressing cells compared to WT-TULP1 expressing cells. The relative band intensity of each protein was normalized to Actin. Data is presented as arbitrary units from two separate western blot densitometry analyses. Statistical significance was calculated using the two-tailed Student’s t -test. * = p
    Xbp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene xbp1
    <t>XBP1</t> regulates NF-κB via ERα signaling. (A) MCF-7 cells were transfected with XBP1 mutants together with the pGL3-Basic or ERE-luc luciferase construct. At 24 h posttransfection, cells were lysed and luciferase activity was measured. (B)
    Xbp1, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc xbp1s
    SubAB induces RIDD in B cells. (A–D) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB for 24 h. The mRNA levels of Hspa5 (A), Pdia4 (B), <t>XBP1s</t> (C), and μS (D) were measured by quantitative RT-PCR. (E) 2-d LPS-stimulated WT and S729A B cells were exposed to 5 µg/ml Tu for 24 h. The μS mRNA levels were measured by quantitative RT-PCR. (F–J) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB for 24 h. The mRNA levels of the indicated genes were measured by quantitative RT-PCR. (K and L) Naive B cells from WT and XBP1 KO mice were stimulated with LPS for 3 d. Some XBP1 KO B cells were incubated with 20 µM B-I09 (K) or 5 µM KIRA6 (L) in the last 24 h of LPS stimulation. The μS mRNA levels were measured by quantitative RT-PCR. Data are shown as means ± SD. Quantitative RT-PCR data are representative of three independent experiments.
    Xbp1s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti xbp1
    CHIKV-induced autophagy is regulated by ER and oxidative stress. (A–C) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of IRE1α (p-IRE1α) and JNK (p-JNK) as well as the formation of spliced form of <t>XBP1</t> (XBP1s) and the conjugation of LC3 (LC3-II). (A) IRE1α and GAPDH were also followed to control protein expression and loading. Similar results were observed in two independent experiments. (B) Immunofluorescence was performed using anti-pIRE1α and anti-E3 antibody. Bars, 15 µm. (C) The number of cells positive for pIRE1α in the E3 + (infected cells) and E3 − (uninfected cells) populations is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (D and E) WT MEFs were pretreated with control siRNA or siRNA against IRE1α for 3 d followed by infection with CHIKV for 24 h at MOI 1. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (F and G) WT MEFs were incubated for 24 h in control media (ø) or in the presence of CHIKV (MOI = 1). (F) Immunofluorescence was performed using an ROS/RNS detection kit that specifically stains oxygen species and free NO. As positive controls for ROS or RNS induction, WT MEFs were incubated for 5 h with pycocyanin and l -arginine, respectively. Bars, 10 µm. (G) Percentage of cells containing ROS or NO among infected with CHIKV and/or pretreated with specific inhibitor of ROS and RNS as indicated is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in two independent experiments. (H) WT MEFs or cells pretreated with siRNA against IRE1α for 3 d were infected with CHIKV for 24 h in presence of a ROS inhibitor. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (I) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of mTOR (p-mTOR), S6KI (p-S6K1), and AMPK (p-AMPK). mTOR, S6K1, AMPK, and GAPDH were also followed to control protein expression and loading. As control to ROS implication, similar experiments were performed in cells pretreated with ROS inducer and/or ROS inhibitor. Black lines indicate that intervening lanes were spliced out. Similar results were observed in three independent experiments. Student’s test: **, P
    Anti Xbp1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Shanghai Genechem xbp1
    CHIKV-induced autophagy is regulated by ER and oxidative stress. (A–C) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of IRE1α (p-IRE1α) and JNK (p-JNK) as well as the formation of spliced form of <t>XBP1</t> (XBP1s) and the conjugation of LC3 (LC3-II). (A) IRE1α and GAPDH were also followed to control protein expression and loading. Similar results were observed in two independent experiments. (B) Immunofluorescence was performed using anti-pIRE1α and anti-E3 antibody. Bars, 15 µm. (C) The number of cells positive for pIRE1α in the E3 + (infected cells) and E3 − (uninfected cells) populations is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (D and E) WT MEFs were pretreated with control siRNA or siRNA against IRE1α for 3 d followed by infection with CHIKV for 24 h at MOI 1. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (F and G) WT MEFs were incubated for 24 h in control media (ø) or in the presence of CHIKV (MOI = 1). (F) Immunofluorescence was performed using an ROS/RNS detection kit that specifically stains oxygen species and free NO. As positive controls for ROS or RNS induction, WT MEFs were incubated for 5 h with pycocyanin and l -arginine, respectively. Bars, 10 µm. (G) Percentage of cells containing ROS or NO among infected with CHIKV and/or pretreated with specific inhibitor of ROS and RNS as indicated is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in two independent experiments. (H) WT MEFs or cells pretreated with siRNA against IRE1α for 3 d were infected with CHIKV for 24 h in presence of a ROS inhibitor. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (I) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of mTOR (p-mTOR), S6KI (p-S6K1), and AMPK (p-AMPK). mTOR, S6K1, AMPK, and GAPDH were also followed to control protein expression and loading. As control to ROS implication, similar experiments were performed in cells pretreated with ROS inducer and/or ROS inhibitor. Black lines indicate that intervening lanes were spliced out. Similar results were observed in three independent experiments. Student’s test: **, P
    Xbp1, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher gene exp xbp1 hs03929085 g1
    BLOC1S1 degradation and <t>XBP1</t> splicing are temporally separate. (A and C) Relative expression of BLOC1S1 in NCI-H929 or RPMI-8226 cells treated with the indicated stressors over time. BLOC1S1 mRNA was measured by SYBR green qPCR and normalized to the GAPDH control and is presented relative to time zero. (B and D) Western blots showing phospho-IRE1, IRE1, and β-actin in the cells treated for panel A. Also shown is agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site from the samples used in panel A. (E to L) TaqMan qPCR measurements of BLOC1S1 , XBP1u , and XBP1s for cells treated as indicated. The data were normalized to the GAPDH control and are presented relative to untreated cells. The data are representative of two independent experiments, and error bars show SEM from three technical replicates.
    Gene Exp Xbp1 Hs03929085 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher gene exp xbp1 mm00457357 m1
    Plasma Cells from ADAM10 Δ/Δ IgG1-cre +/− mice have altered gene expression. ADAM10 Δ/Δ IgG1-cre +/− and controls were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, splenic PCs were isolated via magnetic bead isolation. mRNA was isolated and (A) <t>Xbp1</t> (B) Prdm1 , (C) Irf4 and (D) Bcl6 message levels were determined by qPCR. (E) The ratio of Prdm1 to Bcl6 was calculated. Bars represent the mean ± SE of 3 independent studies; cells from 3 mice from each genotype pooled in each study. (*p
    Gene Exp Xbp1 Mm00457357 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology rabbit anti xbp1
    <t>XBP1</t> mRNA is selectively spliced in NBD rats. RNA extracted from CBLM and HC of PND28 control ( n = 5) and NBD ( n = 7) rats was analyzed by RT-PCR using primers that amplify both unspliced XBP1 (XBP1 U ) and spliced (XBP1 S ) XBP1. (A and B) Representative gel images of XBP1 RT-PCR. Note detection of XBP1 U in control rats and both XBP1 U and XBP1 S in NBD rat CBLM (A) or HC (B). (C and D) PstI digestion of XBP1 RT-PCR products. Note PstI-insensitive XBP1 S band in NBD but not control rat CBLM (C) and HC (D). (E) Sequencing of XBP1 U and XBP1 S from CBLM and HC confirms excision of the 26-nucleotide intron from XBP1 S in NBD rats. (F and G) SYBR Green real-time PCR analysis of total XBP1 mRNA in CBLM and HC of control ( n = 5) and NBD ( n = 7) rats. Note that XBP1 mRNA expression levels are unchanged in NBD CBLM (F) but are significantly increased in NBD HC (G: 1.3-fold increase in NBD; ANOVA, P = 0.005). (H and I) Western blot analysis of XBP1 U (33 kDa) protein levels in CBLM and HC cytoplasmic and nuclear extracts from control ( n = 4) and NBD ( n = 4) rats. Representative blots are shown. (H) Relative band densities for CBLM extracts revealed a trend toward decreased XBP1 U protein in cytoplasmic extracts (2.49-fold decrease in NBD; ANOVA, P = 0.065) and a significant decrease in nuclear extracts (1.72-fold decrease in NBD; ANOVA, P = 0.017) from NBD rats compared to controls. (I) Relative band density for HC extracts revealed significant decreases in XBP1 U protein in both cytoplasmic (2.63-fold decrease in NBD; ANOVA, P = 0.033) and nuclear (3.02-fold decrease in NBD; ANOVA, P = 0.018) extracts from NBD rats compared to results for controls. Asterisks indicate P values of
    Rabbit Anti Xbp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher gene exp xbp1 mm00457359 m1
    Expression of spliced <t>XBP1</t> and its downstream target genes. qRT-PCR data shows significant differences in transcript levels of spliced XBP1 (A) and ERDJ4 (B) in GADD34 -/- mice compared to WT mice at 72 hours post-SCI.
    Gene Exp Xbp1 Mm00457359 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti xbp 1 antibody
    XBP-1S and XBP-1U interact with Tax. (A) Co-IP was performed with the indicated antibody (i.e., <t>anti-XBP-1</t> or anti-Tax antibody) and cell lysates (CL) prepared from HEK293 cells transfected with XBP-1S/Tax or XBP-1U/Tax expression vectors. Normal rabbit IgG was used as a negative control. The expression of XBP-1S, XBP-1U, and Tax was confirmed by Western blotting (i.e., CL input). In vitro-translated XBP-1S and XBP-1U (i.e., TNT) were run on the same gel for comparison. The immunoprecipitated complexes were analyzed by Western blotting.
    Anti Xbp 1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gene exp xbp1 mm03464496 m1
    Effect of nsPEF on the activation of the endoplasmic reticulum (ER) stress sensors IRE1 ( A ) and PERK ( B ). EL-4 cells (top panels) and CT26 cell (bottom panels) were treated with iso-effective doses of 100 and 300 pulses, respectively (200 ns, 7 kV/cm, 10 Hz). Samples were collected at 5 h post treatment. In (A) the expression level of <t>XBP1</t> in both EL-4 and CT26 was measured by real-time quantitative PCR. The gene mRNA level was normalized to the housekeeping HPRT gene mRNA and is shown as relative expression. In ( B ) phosphorylation of eIF2α was measured by Western blot using an anti-phospho-eIF2α (Serine 51) antibody. Left panels show a representative image for both EL-4 (top panel) and CT26 cells (bottom panel) with eIF2α (phosphorylated and total) and the housekeeping Vinculin protein seen as a 38 and 140 kDa band, respectively. Graphs on the right are the quantifications of the p-eIF2α expressed as fold to sham. 1 µM thaspigargin (Thaps.) was used as a positive control for ER stress induction. Mean +/− s.e. n = 3 for both A and B. * p
    Gene Exp Xbp1 Mm03464496 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    xbp1s  (Abcam)
    91
    Abcam xbp1s
    Sodium demethylcantharidate induced ER stress in HHC cells. ER stress markers, including p-IRE1, GRP78/BiP, <t>XBP1s,</t> Caspase 12 and CHOP were analyzed in concentration course manner by Western blotting.
    Xbp1s, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GenWay xbp1 s
    Knockdown of ERα inhibits ICI-induced UPR signaling. LCC1 and -9 cells were transfected with ERα shRNA or control pcDNA and treated with or without 100 nM ICI for 72 h. A–C ) Western blot hybridization of treated protein homogenates was used to measure GRP78 ( A ), <t>XBP1-S</t> ( B ), and CHOP ( C ) expression. D ) XBP1 and ERE activity in LCC9 cells was determined by using a CRE-luc system ( n = 3). * P
    Xbp1 S, supplied by GenWay, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The 4‐phenylbutyrate (4‐PBA) reduced tunicamaycin‐induced resistin and ER stress markers messenger ribonucleic acid (mRNA) in THP‐1 human monocytes. THP‐1 cells were pre‐incubated with 750, 1,500, 3,000 or 7,500 μmol/L of 4‐PBA, a chemical chaperone, for 14 h, and 0.1 μg/mL tunicamycin was subsequently added to the medium for 24 h. The mRNA of (a) endoplasmic reticulum stress markers (immunoglobulin heavy chain‐binding protein [BiP], pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase [PERK], activating transcription factor 4 [ATF4], CAAT/enhancer binding protein‐α homologous protein [CHOP], ATF6, inositol requiring enzyme 1 [IRE1] and X‐box binding protein 1 [XBP1]) and (b) resistin ( RETN ) were quantitated using reverse transcription polymerase chain reaction and shown relative to 18S rRNA mRNA as described in the Materials and Methods. The mRNA of control (4‐PBA [–], tunicamycin [–]) was defined as 1.0. The mean ± standard error of the mean of five separate experiments with triplicate samples is shown. anova ; P = 8.49 × 10 −8 (BiP), P = 0.00040 (PERK), P = 2.46 × 10 −6 (ATF4), P = 1.53 × 10 −5 (CHOP), P = 0.00085 (ATF6), P = 0.0070 (IRE1), P = 0.00016 (XBP1), P = 5.44 × 10 −6 (RETN); Scheffe's test; †† P

    Journal: Journal of Diabetes Investigation

    Article Title: Endoplasmic reticulum stress induced by tunicamycin increases resistin messenger ribonucleic acid through the pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase–activating transcription factor 4–CAAT/enhancer binding protein‐α homologous protein pathway in THP‐1 human monocytes

    doi: 10.1111/jdi.12434

    Figure Lengend Snippet: The 4‐phenylbutyrate (4‐PBA) reduced tunicamaycin‐induced resistin and ER stress markers messenger ribonucleic acid (mRNA) in THP‐1 human monocytes. THP‐1 cells were pre‐incubated with 750, 1,500, 3,000 or 7,500 μmol/L of 4‐PBA, a chemical chaperone, for 14 h, and 0.1 μg/mL tunicamycin was subsequently added to the medium for 24 h. The mRNA of (a) endoplasmic reticulum stress markers (immunoglobulin heavy chain‐binding protein [BiP], pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase [PERK], activating transcription factor 4 [ATF4], CAAT/enhancer binding protein‐α homologous protein [CHOP], ATF6, inositol requiring enzyme 1 [IRE1] and X‐box binding protein 1 [XBP1]) and (b) resistin ( RETN ) were quantitated using reverse transcription polymerase chain reaction and shown relative to 18S rRNA mRNA as described in the Materials and Methods. The mRNA of control (4‐PBA [–], tunicamycin [–]) was defined as 1.0. The mean ± standard error of the mean of five separate experiments with triplicate samples is shown. anova ; P = 8.49 × 10 −8 (BiP), P = 0.00040 (PERK), P = 2.46 × 10 −6 (ATF4), P = 1.53 × 10 −5 (CHOP), P = 0.00085 (ATF6), P = 0.0070 (IRE1), P = 0.00016 (XBP1), P = 5.44 × 10 −6 (RETN); Scheffe's test; †† P

    Article Snippet: The following primers were purchased from Applied Biosystems and used for RT–PCR: glyceraldehyde 3‐phosphate dehydrogenase (Hs99999905_m1), 18S (Hs99999901_s1), RETN (Hs00220767_m1), BiP/HSPA5 (Hs00607129_gH), PERK/EIF2AK3 (Hs00984005_m1), ATF4 (Hs00909569_g1), CHOP/DDIT3 (Hs00358796_g1), ATF6α (Hs00232586_m1), IRE1α/ERN1 (Hs00176385_m1) and XBP1 (Hs00231936_m1).

    Techniques: Incubation, Binding Assay, Reverse Transcription Polymerase Chain Reaction

    Integration of ChIPmentation and RNA-seq data reveals XBP1 direct target genes and its regulatory network. a Venn diagram comparing previously reported XBP1 target genes of other secretory cell types with the Th2 direct target genes of this study. XBP1 direct target genes of this study are those that are common in both “XBP1-occupied genes in Th2” and “Differentially expressed genes (Th2 → Th2+4μ8c)” categories. The XBP1 direct target genes of B cell/plasma cell, skeletal muscle cells, and pancreatic β-cells were as observed by Acosta-Alvear et al. [ 17 ] and have been used here for comparison. b Heatmap showing the pattern of XBP1 direct target gene expression. The top 38 genes that follow a distinct pattern have been displayed. c Transcriptional regulatory network: transcription factors that are direct target of XBP1. The genes in the network are differentially expressed (upregulated—red; downregulated—blue) up on 4μ8c treatment. The transcription factors that are not differentially expressed but have a XBP1 ChIPseq peak are shown in the right-hand side list

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: Integration of ChIPmentation and RNA-seq data reveals XBP1 direct target genes and its regulatory network. a Venn diagram comparing previously reported XBP1 target genes of other secretory cell types with the Th2 direct target genes of this study. XBP1 direct target genes of this study are those that are common in both “XBP1-occupied genes in Th2” and “Differentially expressed genes (Th2 → Th2+4μ8c)” categories. The XBP1 direct target genes of B cell/plasma cell, skeletal muscle cells, and pancreatic β-cells were as observed by Acosta-Alvear et al. [ 17 ] and have been used here for comparison. b Heatmap showing the pattern of XBP1 direct target gene expression. The top 38 genes that follow a distinct pattern have been displayed. c Transcriptional regulatory network: transcription factors that are direct target of XBP1. The genes in the network are differentially expressed (upregulated—red; downregulated—blue) up on 4μ8c treatment. The transcription factors that are not differentially expressed but have a XBP1 ChIPseq peak are shown in the right-hand side list

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: RNA Sequencing Assay, Expressing

    IRE1a inhibition delays cell cycle progression through the S and G2/M phase. a Left panel: heatmap of differentially expressed cell cycle stage-associated genes in the 4μ8c-treated and untreated Th2 transcriptome. Right panel: heatmap of XBP1 direct target genes that are known to be involved in cell cycling. The heatmap shows scaled expression values denoted as row Z -score, in red-blue color scale with red indicating increased expression and blue indicating decreased expression. b Cell cycle analysis of Th2 lymphocytes after 72 h of activation, using FUCCI mouse line that express mCherry-tagged CDT1 and Venus-tagged GEMININ. Upper left: diagrammatic representation of cell cycle stages in used FUCCI mouse. Upper right: comparison of cells (% of total) obtained from different stages of cell cycle in Th2 and 4μ8c-treated Th2 ( n = 6). Lower panels: one representative FACS profile of Th2 and 4μ8c-treated Th2 showing CDT1 and GEMININ expressing cells

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: IRE1a inhibition delays cell cycle progression through the S and G2/M phase. a Left panel: heatmap of differentially expressed cell cycle stage-associated genes in the 4μ8c-treated and untreated Th2 transcriptome. Right panel: heatmap of XBP1 direct target genes that are known to be involved in cell cycling. The heatmap shows scaled expression values denoted as row Z -score, in red-blue color scale with red indicating increased expression and blue indicating decreased expression. b Cell cycle analysis of Th2 lymphocytes after 72 h of activation, using FUCCI mouse line that express mCherry-tagged CDT1 and Venus-tagged GEMININ. Upper left: diagrammatic representation of cell cycle stages in used FUCCI mouse. Upper right: comparison of cells (% of total) obtained from different stages of cell cycle in Th2 and 4μ8c-treated Th2 ( n = 6). Lower panels: one representative FACS profile of Th2 and 4μ8c-treated Th2 showing CDT1 and GEMININ expressing cells

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: Inhibition, Expressing, Cell Cycle Assay, Activation Assay, FACS

    IRE1a-XBP1 pathway is required for cytokine expression and secretion in Th2 lymphocyte. Naïve T helper cells were cultured following Th2 activation condition in the presence of IRE1a inhibitor 4μ8c for 3 days, rested for 2 days, reactivated by coated plate, and analyzed by flow cytometry to detect intra-cellular cytokines IL4, IL5, and IL13 expression. Representative FACS profiles are displayed in the first two columns. The intra-cellular cytokine expression is compared in column 3, with three to seven independent biological replicates. Fourth column: cell culture supernatants from 4μ8c-treated or DMSO-treated Th2 were analyzed by ELISA to measure the cytokine concentration. FACS gating: lymphocytes > singlets > live cells > cytokines

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: IRE1a-XBP1 pathway is required for cytokine expression and secretion in Th2 lymphocyte. Naïve T helper cells were cultured following Th2 activation condition in the presence of IRE1a inhibitor 4μ8c for 3 days, rested for 2 days, reactivated by coated plate, and analyzed by flow cytometry to detect intra-cellular cytokines IL4, IL5, and IL13 expression. Representative FACS profiles are displayed in the first two columns. The intra-cellular cytokine expression is compared in column 3, with three to seven independent biological replicates. Fourth column: cell culture supernatants from 4μ8c-treated or DMSO-treated Th2 were analyzed by ELISA to measure the cytokine concentration. FACS gating: lymphocytes > singlets > live cells > cytokines

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: Expressing, Cell Culture, Activation Assay, Flow Cytometry, Cytometry, FACS, Enzyme-linked Immunosorbent Assay, Concentration Assay

    IRE1a-XBP1 pathway promotes activation-dependent Th2 cell proliferation and cell cycling. a Left panel: hierarchical clustering of differentially expressed cell proliferation-associated genes in the 4μ8c-treated and untreated Th2 transcriptome. Right panel: hierarchical clustering of XBP1 direct target genes that are known to be involved in cell proliferation. The heatmap shows scaled expression values denoted as row Z -score, in red-blue color scale with red indicating increased expression and blue indicating decreased expression. b Splenic naïve T helper cells were stained with CellTrace Violet dye and activated for 72 h under Th2 differentiation conditions and analyzed by flow cytometry. Generations of Th2 cells are in “red” and 4μ8c-treated cells are in “blue” in the histogram of cell proliferation (left panel, one representative experiment). Graphical representation of division index as obtained from five independent biological replicates (right panel)

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: IRE1a-XBP1 pathway promotes activation-dependent Th2 cell proliferation and cell cycling. a Left panel: hierarchical clustering of differentially expressed cell proliferation-associated genes in the 4μ8c-treated and untreated Th2 transcriptome. Right panel: hierarchical clustering of XBP1 direct target genes that are known to be involved in cell proliferation. The heatmap shows scaled expression values denoted as row Z -score, in red-blue color scale with red indicating increased expression and blue indicating decreased expression. b Splenic naïve T helper cells were stained with CellTrace Violet dye and activated for 72 h under Th2 differentiation conditions and analyzed by flow cytometry. Generations of Th2 cells are in “red” and 4μ8c-treated cells are in “blue” in the histogram of cell proliferation (left panel, one representative experiment). Graphical representation of division index as obtained from five independent biological replicates (right panel)

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: Activation Assay, Expressing, Staining, Flow Cytometry, Cytometry

    T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: Activation Assay, Transformation Assay, Purification, Selection, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Staining, FACS

    T helper cells upregulate IRE1a-XBP1 pathway in vivo during infection. Splenocytes from nematode ( Nippostrongylus brasiliensis )-infected mouse (7 days post-infection) were stained with a PE-conjugated anti-XBP1s antibody and analyzed by flow cytometry (gating strategy: singlet > live cells > CD4 + CD3e + > XBP1s + ). One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 4) is shown in the “right panel”

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: T helper cells upregulate IRE1a-XBP1 pathway in vivo during infection. Splenocytes from nematode ( Nippostrongylus brasiliensis )-infected mouse (7 days post-infection) were stained with a PE-conjugated anti-XBP1s antibody and analyzed by flow cytometry (gating strategy: singlet > live cells > CD4 + CD3e + > XBP1s + ). One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 4) is shown in the “right panel”

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: In Vivo, Infection, Staining, Flow Cytometry, Cytometry, FACS

    Genome-wide chromatin occupancy of XBP1 transcription factor in Th2 lymphocyte . XBP1 ChIPmentation was performed in in vitro differentiated Th2 cells to obtain genome-wide XBP1 chromatin occupancy. a Snapshot of XBP1 binding peaks around indicated representative genes from the UCSC genome browser. b Genomic distribution of XBP1 binding peaks. The sector corresponding to the promoter includes sequences up to 1 kb upstream and 100 bp downstream from the TSS. c Comparing the XBP1 motifs from the JASPAR database (top), ChIP-seq of the human breast cancer cell lines (middle), and mouse Th2 lymphocytes (bottom). d Motif frequencies of XPB1 and NF-Y around the binding peaks of XBP1. e Biological processes’ GO terms enriched within XBP1 binding peaks analyzed by GREAT

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: Genome-wide chromatin occupancy of XBP1 transcription factor in Th2 lymphocyte . XBP1 ChIPmentation was performed in in vitro differentiated Th2 cells to obtain genome-wide XBP1 chromatin occupancy. a Snapshot of XBP1 binding peaks around indicated representative genes from the UCSC genome browser. b Genomic distribution of XBP1 binding peaks. The sector corresponding to the promoter includes sequences up to 1 kb upstream and 100 bp downstream from the TSS. c Comparing the XBP1 motifs from the JASPAR database (top), ChIP-seq of the human breast cancer cell lines (middle), and mouse Th2 lymphocytes (bottom). d Motif frequencies of XPB1 and NF-Y around the binding peaks of XBP1. e Biological processes’ GO terms enriched within XBP1 binding peaks analyzed by GREAT

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: Genome Wide, In Vitro, Binding Assay, Chromatin Immunoprecipitation

    Differential gene expression in Th2 due to the inhibition of IRE1a-XBP1 by 4μ8c. Naïve T helper cells were activated under Th2 differentiation conditions in the presence or absence of 4μ8c. Cells were activated in anti-CD3e and anti-CD28 antibody-coated plates for 3 days, rested for 2 days, and reactivated in coated plates for 6 h. The RNAseq data were analyzed for differential gene expression. a Venn diagram showing the numbers of differentially expressed genes in different experimental conditions. “Naïve → Th2” indicates the differentially expressed genes between naïve T helpers and Th2 cells. “Th2 → Th2+4μ8c” indicates the differentially expressed genes between untreated and 4μ8c-treated Th2. b Heatmap showing differentially expressed genes that are well known to be involved in resolution of ER stress imposed by unfolded protein response. The heatmap shows scaled expression values denoted as row Z -score, in red-blue color scale with red indicating increased expression and blue indicating decreased expression. c Gene ontology (GO) analysis of the differentially expressed genes between Th2 and 4μ8c-treated Th2

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: Differential gene expression in Th2 due to the inhibition of IRE1a-XBP1 by 4μ8c. Naïve T helper cells were activated under Th2 differentiation conditions in the presence or absence of 4μ8c. Cells were activated in anti-CD3e and anti-CD28 antibody-coated plates for 3 days, rested for 2 days, and reactivated in coated plates for 6 h. The RNAseq data were analyzed for differential gene expression. a Venn diagram showing the numbers of differentially expressed genes in different experimental conditions. “Naïve → Th2” indicates the differentially expressed genes between naïve T helpers and Th2 cells. “Th2 → Th2+4μ8c” indicates the differentially expressed genes between untreated and 4μ8c-treated Th2. b Heatmap showing differentially expressed genes that are well known to be involved in resolution of ER stress imposed by unfolded protein response. The heatmap shows scaled expression values denoted as row Z -score, in red-blue color scale with red indicating increased expression and blue indicating decreased expression. c Gene ontology (GO) analysis of the differentially expressed genes between Th2 and 4μ8c-treated Th2

    Article Snippet: As expected, binding peaks were identified around promoter regions in known XBP1 target genes, such as Hspa5 that encodes ER-chaperone protein BiP also known as Grp78; a binding event was also observed around the promoter of XBP1 itself (Fig. ), indicating potential auto-regulation of XBP1.

    Techniques: Expressing, Inhibition

    Detection of endogenous XBP-1 in mouse oocytes and preimplantation embryos in vivo . A. A specific anti-XBP-1 antibody was used to detect localization of XBP-1 in mouse oocytes via immunostaining (green). Nuclei were stained with DAPI (blue); Scale bar, 20 µm. B. Confocal immunofluorescence images of mouse pre-implantation embryos. The XBP-1 protein was detected using a specific antibody (green). Negative control embryos were probed directly with the secondary antibody. Nuclei were stained with DAPI (blue); Scale bar, 20 μm.

    Journal: PLoS ONE

    Article Title: Inhibition of Endoplasmic Reticulum Stress Improves Mouse Embryo Development

    doi: 10.1371/journal.pone.0040433

    Figure Lengend Snippet: Detection of endogenous XBP-1 in mouse oocytes and preimplantation embryos in vivo . A. A specific anti-XBP-1 antibody was used to detect localization of XBP-1 in mouse oocytes via immunostaining (green). Nuclei were stained with DAPI (blue); Scale bar, 20 µm. B. Confocal immunofluorescence images of mouse pre-implantation embryos. The XBP-1 protein was detected using a specific antibody (green). Negative control embryos were probed directly with the secondary antibody. Nuclei were stained with DAPI (blue); Scale bar, 20 μm.

    Article Snippet: In previous studies, XBP-1 was shown to be essential for mouse placental development .

    Techniques: In Vivo, Immunostaining, Staining, Immunofluorescence, Negative Control

    Induction of active XBP-1 in one-cell stage embryos. A. XBP-1 mRNA was spliced to produce the spliced and unspliced forms in the presence of TM and sorbitol. B. Immunofluorescence micrographs of two-cell stage embryos with TM or sorbitol. Active XBP - 1 protein was detected in nuclei in the presence or absence of stress inducers (green). Negative control embryos were probed directly with the secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. C. Active and inactive XBP-1 proteins were detected in the presence and absence of stress inducers using Western blotting. β-actin served as a control. D. Quantification of the Western blot analysis in C. The data were presented as means ± SD from three independent experiments.

    Journal: PLoS ONE

    Article Title: Inhibition of Endoplasmic Reticulum Stress Improves Mouse Embryo Development

    doi: 10.1371/journal.pone.0040433

    Figure Lengend Snippet: Induction of active XBP-1 in one-cell stage embryos. A. XBP-1 mRNA was spliced to produce the spliced and unspliced forms in the presence of TM and sorbitol. B. Immunofluorescence micrographs of two-cell stage embryos with TM or sorbitol. Active XBP - 1 protein was detected in nuclei in the presence or absence of stress inducers (green). Negative control embryos were probed directly with the secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. C. Active and inactive XBP-1 proteins were detected in the presence and absence of stress inducers using Western blotting. β-actin served as a control. D. Quantification of the Western blot analysis in C. The data were presented as means ± SD from three independent experiments.

    Article Snippet: In previous studies, XBP-1 was shown to be essential for mouse placental development .

    Techniques: Immunofluorescence, Negative Control, Staining, Western Blot

    Detection of XBP-1 splicing in mouse preimplantation embryos. A. Expression of XBP-1 mRNA was analyzed using RT-PCR. RNA was isolated from 50 embryos of each stage and reverse-transcribed. cDNA was used as the template for PCR. XBP-1 s and XBP-1 u amplicons were separated on a 2% (w/v) agarose gel. B. Expression patterns of active and inactive XBP-1 proteins were detected in two-cell embryos using Western blotting. β-actin served as the control. C. Quantification of the Western blot analysis in B. The data were presented as means ± SD from three independent experiments.

    Journal: PLoS ONE

    Article Title: Inhibition of Endoplasmic Reticulum Stress Improves Mouse Embryo Development

    doi: 10.1371/journal.pone.0040433

    Figure Lengend Snippet: Detection of XBP-1 splicing in mouse preimplantation embryos. A. Expression of XBP-1 mRNA was analyzed using RT-PCR. RNA was isolated from 50 embryos of each stage and reverse-transcribed. cDNA was used as the template for PCR. XBP-1 s and XBP-1 u amplicons were separated on a 2% (w/v) agarose gel. B. Expression patterns of active and inactive XBP-1 proteins were detected in two-cell embryos using Western blotting. β-actin served as the control. C. Quantification of the Western blot analysis in B. The data were presented as means ± SD from three independent experiments.

    Article Snippet: In previous studies, XBP-1 was shown to be essential for mouse placental development .

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

    Dose-dependent effects of stress inducers on localization of XBP-1. A. Two-cell stage embryos cultured with activator or inhibitor of ER stress were examined using a specific anti-XBP-1 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. B. Active XBP-1 in two-cell embryos in the presence of activator or inhibitor of ER stress was analyzed using Western blotting. β-actin served as the control. Each experiment was repeated three times. (*) indicates a statistically significant difference from control (P

    Journal: PLoS ONE

    Article Title: Inhibition of Endoplasmic Reticulum Stress Improves Mouse Embryo Development

    doi: 10.1371/journal.pone.0040433

    Figure Lengend Snippet: Dose-dependent effects of stress inducers on localization of XBP-1. A. Two-cell stage embryos cultured with activator or inhibitor of ER stress were examined using a specific anti-XBP-1 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. B. Active XBP-1 in two-cell embryos in the presence of activator or inhibitor of ER stress was analyzed using Western blotting. β-actin served as the control. Each experiment was repeated three times. (*) indicates a statistically significant difference from control (P

    Article Snippet: In previous studies, XBP-1 was shown to be essential for mouse placental development .

    Techniques: Cell Culture, Staining, Western Blot

    ZIKV infection induces the splicing of xbp1 and the translocation of XBP1s into the nuclei of human neural cells. a , b CCF- STTG1 and SK-N-SH were infected with ZIKV at an MOI of 5 PFU/cell. Positive control (Tm) was treated with 2 μM tunicamycin. RNA was extracted at 24 and 48 hpi and the first strand cDNA was synthesized. Primers that specifically amplified xbp1s and total xbp1 (including both xbp1u and xbp1s ) were used to analyze xbp1s via RT-qPCR. The relative expression levels of the xbp1s and t-xbp1 were calculated according to the 2 −∆∆ Ct method. The ratios of xbp1s / t-xbp1 between mock-infected and ZIKV-infected were calculated. Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Journal: Journal of Neuroinflammation

    Article Title: ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells

    doi: 10.1186/s12974-018-1311-5

    Figure Lengend Snippet: ZIKV infection induces the splicing of xbp1 and the translocation of XBP1s into the nuclei of human neural cells. a , b CCF- STTG1 and SK-N-SH were infected with ZIKV at an MOI of 5 PFU/cell. Positive control (Tm) was treated with 2 μM tunicamycin. RNA was extracted at 24 and 48 hpi and the first strand cDNA was synthesized. Primers that specifically amplified xbp1s and total xbp1 (including both xbp1u and xbp1s ) were used to analyze xbp1s via RT-qPCR. The relative expression levels of the xbp1s and t-xbp1 were calculated according to the 2 −∆∆ Ct method. The ratios of xbp1s / t-xbp1 between mock-infected and ZIKV-infected were calculated. Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Article Snippet: It implied that ZIKV infection induced the splicing of xbp1 .

    Techniques: Infection, Translocation Assay, Positive Control, Synthesized, Amplification, Quantitative RT-PCR, Expressing

    ZIKV infects the nervous tissues of the mouse brain and activates the ER stress markers . Three-week-old AG6 mice were infected with 1 × 10 5 PFU/mouse (each dose, N = 3) via intraperitoneal injection. a . The total mRNA of the mouse brain was extracted, and ZIKV mRNA was detected at 2 dpi and 5 dpi via RT-qPCR. b , c The total lysates of mouse brain were analyzed at 5 dpi via western blot. An equal amount of lysates were analyzed with anti-ZIKV envelope protein antibody ( b ), anti-BIP antibody, anti-ATF6n antibody, anti-phoshpo-IRE1 antibody, and anti-XBP1 antibody ( c ). d . The band was analyzed by using Image Lab 4.0.1. The fold change of the target protein / β-actin was calculated. The protein expression of mock-infected and ZIKV-infected samples were normalized with the internal control β-actin. e . Primers that specifically amplified bip , ire1 , atf6 , t-xbp1 , and xbp1s were used. The relative expression levels of the genes were calculated according to the 2 −∆∆ Ct method. Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Journal: Journal of Neuroinflammation

    Article Title: ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells

    doi: 10.1186/s12974-018-1311-5

    Figure Lengend Snippet: ZIKV infects the nervous tissues of the mouse brain and activates the ER stress markers . Three-week-old AG6 mice were infected with 1 × 10 5 PFU/mouse (each dose, N = 3) via intraperitoneal injection. a . The total mRNA of the mouse brain was extracted, and ZIKV mRNA was detected at 2 dpi and 5 dpi via RT-qPCR. b , c The total lysates of mouse brain were analyzed at 5 dpi via western blot. An equal amount of lysates were analyzed with anti-ZIKV envelope protein antibody ( b ), anti-BIP antibody, anti-ATF6n antibody, anti-phoshpo-IRE1 antibody, and anti-XBP1 antibody ( c ). d . The band was analyzed by using Image Lab 4.0.1. The fold change of the target protein / β-actin was calculated. The protein expression of mock-infected and ZIKV-infected samples were normalized with the internal control β-actin. e . Primers that specifically amplified bip , ire1 , atf6 , t-xbp1 , and xbp1s were used. The relative expression levels of the genes were calculated according to the 2 −∆∆ Ct method. Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Article Snippet: It implied that ZIKV infection induced the splicing of xbp1 .

    Techniques: Mouse Assay, Infection, Injection, Quantitative RT-PCR, Western Blot, Expressing, Amplification

    ZIKV infection upregulates the expression of phospho-IRE1, XBP1, and ATF6 in the mouse brain. Three-week-old AG6 mice were infected with 1 × 10 5 PFU/mouse (each dose, N = 3) via intraperitoneal injection. a , b , c Sections of the cerebrum, cerebellum, and mesocephalon were obtained at 5 dpi, and anti-phospho-IRE1 antibody ( a ), anti-XBP1 antibody (recognizing both non-spliced and spliced isoforms of XBP1) ( b ), and anti-ATF6 antibody (recognizing both cleaved and full forms of ATF6) ( c ) were used for immunohistochemistry assay. Purple dots represent the cell nucleus. Brown spots represent the ZIKV envelope protein. A representative of three independent experiments is shown. Positive cells marked by white squares were magnified nine times and showed on the top-right corner. d , e , f Aperio ImageScope viewing software was used to analyze the percentage of positivity (algorithm, positive pixel count). Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Journal: Journal of Neuroinflammation

    Article Title: ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells

    doi: 10.1186/s12974-018-1311-5

    Figure Lengend Snippet: ZIKV infection upregulates the expression of phospho-IRE1, XBP1, and ATF6 in the mouse brain. Three-week-old AG6 mice were infected with 1 × 10 5 PFU/mouse (each dose, N = 3) via intraperitoneal injection. a , b , c Sections of the cerebrum, cerebellum, and mesocephalon were obtained at 5 dpi, and anti-phospho-IRE1 antibody ( a ), anti-XBP1 antibody (recognizing both non-spliced and spliced isoforms of XBP1) ( b ), and anti-ATF6 antibody (recognizing both cleaved and full forms of ATF6) ( c ) were used for immunohistochemistry assay. Purple dots represent the cell nucleus. Brown spots represent the ZIKV envelope protein. A representative of three independent experiments is shown. Positive cells marked by white squares were magnified nine times and showed on the top-right corner. d , e , f Aperio ImageScope viewing software was used to analyze the percentage of positivity (algorithm, positive pixel count). Data represented three independent experiments and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Article Snippet: It implied that ZIKV infection induced the splicing of xbp1 .

    Techniques: Infection, Expressing, Mouse Assay, Injection, Immunohistochemistry, Software

    ZIKV infection stimulates ATF6n and XBP1s to translocate into the cell nuclei of neural cells in the mouse brain. Three-week-old AG6 mice were infected with 1 × 10 5 PFU/mouse (each dose, N = 3) via intraperitoneal injection. a , b The whole brain sections of AG6 mice were stained with DAPI (blue), anti-ZIKV envelope protein antibody (red), and anti-XBP1 ( a ) or anti-ATF6 ( b ) antibody (green) and were used for immunohistofluorescence assay at 5 dpi. Images of the whole brain were captured by using a digital slice scanning analysis system (Pannoramic MIDI). A representative sub-area of mesocephalon is shown. Positive cells marked by white squares were magnified nine times and shown on the top-right corner. c Positive rates of nuclear localization were counted. Data represent three area of mesocephalon through random selection and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Journal: Journal of Neuroinflammation

    Article Title: ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells

    doi: 10.1186/s12974-018-1311-5

    Figure Lengend Snippet: ZIKV infection stimulates ATF6n and XBP1s to translocate into the cell nuclei of neural cells in the mouse brain. Three-week-old AG6 mice were infected with 1 × 10 5 PFU/mouse (each dose, N = 3) via intraperitoneal injection. a , b The whole brain sections of AG6 mice were stained with DAPI (blue), anti-ZIKV envelope protein antibody (red), and anti-XBP1 ( a ) or anti-ATF6 ( b ) antibody (green) and were used for immunohistofluorescence assay at 5 dpi. Images of the whole brain were captured by using a digital slice scanning analysis system (Pannoramic MIDI). A representative sub-area of mesocephalon is shown. Positive cells marked by white squares were magnified nine times and shown on the top-right corner. c Positive rates of nuclear localization were counted. Data represent three area of mesocephalon through random selection and error bars indicate mean ± SD. Statistical analyses were performed using multiple t tests ( N = 3) ( P

    Article Snippet: It implied that ZIKV infection induced the splicing of xbp1 .

    Techniques: Infection, Mouse Assay, Injection, Staining, Immunohistofluorescence, Selection

    Effects of UDCA on OSS-induced ER stress in endothelial cells. HUVECs were cultured under static, LSS, or OSS conditions for the indicated time with or without UDCA (100 μM). The expression levels of ER stress markers, including XBP-1 and CHOP, were detected by Western blotting. (A, B) Expressions of ER stress markers in endothelial cells under fluid conditions. * P

    Journal: Molecules and Cells

    Article Title: Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting Endoplasmic Reticulum (ER) Stress Induced by Disturbed Flow

    doi: 10.14348/molcells.2015.0094

    Figure Lengend Snippet: Effects of UDCA on OSS-induced ER stress in endothelial cells. HUVECs were cultured under static, LSS, or OSS conditions for the indicated time with or without UDCA (100 μM). The expression levels of ER stress markers, including XBP-1 and CHOP, were detected by Western blotting. (A, B) Expressions of ER stress markers in endothelial cells under fluid conditions. * P

    Article Snippet: ER stress levels were measured by quantifying the classical markers of the UPR pathway, including phospho-PERK, phospho-eIF2α, XBP-1, p50 ATF6, and CHOP.

    Techniques: Cell Culture, Expressing, Western Blot

    Hcy induces ER stress and inflammation and upregulates Ero1α in the thoracic aortas of HHcy mice. (A) The lysates of the thoracic aortas in normal (Ctrl) and HHcy mice were subjected to immunoblotting analysis for spliced XBP1 (XBP1s), unspliced XBP1 (XBP1u), phosphorylated eIF2α (p-eIF2α), and eIF2α. Representative blots were shown. Statistical analysis for XBP1s/XBP1u and p-eIF2α/eIF2α was indicated. Data were presented as mean ± SEM from seven biological replicates, **p

    Journal: Redox Biology

    Article Title: Homocysteine causes vascular endothelial dysfunction by disrupting endoplasmic reticulum redox homeostasis

    doi: 10.1016/j.redox.2018.09.021

    Figure Lengend Snippet: Hcy induces ER stress and inflammation and upregulates Ero1α in the thoracic aortas of HHcy mice. (A) The lysates of the thoracic aortas in normal (Ctrl) and HHcy mice were subjected to immunoblotting analysis for spliced XBP1 (XBP1s), unspliced XBP1 (XBP1u), phosphorylated eIF2α (p-eIF2α), and eIF2α. Representative blots were shown. Statistical analysis for XBP1s/XBP1u and p-eIF2α/eIF2α was indicated. Data were presented as mean ± SEM from seven biological replicates, **p

    Article Snippet: The following antibodies were used in this work: anti-GPx7 (Abclonal, A3902), anti-NRF2 (Abcam, ab62352), anti-BiP (Sigma, G8918), anti-Ero1α (Merck Millipore, MABT376), anti-XBP1 (Santa Cruz, sc-7160), anti-p-eIF2α (Cell Signaling Technology, D9G8), anti-eIF2α (Abclonal, D7D3), anti-CHOP (Cell Signaling Technology, L63F7), anti-GAPDH (Sigma-Aldrich, G9295), anti-β-actin (Sigma-Aldrich, A3854), anti-ICAM-1 (Santa Cruz, sc-7891), anti-VCAM-1 (Santa Cruz, sc-8304), anti-α-tubulin (Sigma-Aldrich, T6074), anti-Lamin B1 (Abcam, ab16048), anti-HIF-1α (Novusbio, NB100-105), anti-dimedone (Merck Millipore, ABS30), anti-calreticulin (Abcam, ab2907), anti-PDI (Abcam, ab2792), non-specific mouse IgG (Beyotime, A7028), goat anti-rabbit IgG-peroxidase (Sigma-Aldrich, A0545), goat anti-mouse IgG-peroxidase (Sigma-Aldrich, A4416), goat anti-rabbit Alexa Fluor 488 (Invitrogen, A11034), goat anti-mouse Alexa Fluor 568 (Invitrogen, A11004), goat anti-rabbit Alexa Fluor 568 (Invitrogen, A21069), and goat anti-mouse Alexa Fluor 647 (Invitrogen, A31571).

    Techniques: Mouse Assay

    GCN5 overexpression hampers UPR in vivo (A) 293T cells were treated with 10 μg/ml Tm for the indicated time. Expression of GCN5, PCAF, BiP, and actin were analyzed by Western blotting. (B) Cells were transfected with a mock or a GCN5 expression vectors, followed by Tm (10 μg/ml, 16 hrs) or Tg (300 nM, 4 hrs) treatment. Both Tm and Tg were dissolved in DMSO and the final concentration of DMSO in the culture was kept at 0.1%. Cells treated with 0.1% DMSO were served as a negative control. Expression of endogenous BiP, CHOP, EDEM, XBP-1, and ERdj4 genes was determined by qRT-PCR. Cells were transfected with an empty or a GCN5 plasmid followed by Tm (C) or Tg (D) treatment. ChIP-quantitative-PCR assays were carried out to quantify the abundance of XBP-1S located on the promoters of BiP, CHOP, and EDEM genes. * P

    Journal: Oncotarget

    Article Title: GCN5 inhibits XBP-1S-mediated transcription by antagonizing PCAF action

    doi:

    Figure Lengend Snippet: GCN5 overexpression hampers UPR in vivo (A) 293T cells were treated with 10 μg/ml Tm for the indicated time. Expression of GCN5, PCAF, BiP, and actin were analyzed by Western blotting. (B) Cells were transfected with a mock or a GCN5 expression vectors, followed by Tm (10 μg/ml, 16 hrs) or Tg (300 nM, 4 hrs) treatment. Both Tm and Tg were dissolved in DMSO and the final concentration of DMSO in the culture was kept at 0.1%. Cells treated with 0.1% DMSO were served as a negative control. Expression of endogenous BiP, CHOP, EDEM, XBP-1, and ERdj4 genes was determined by qRT-PCR. Cells were transfected with an empty or a GCN5 plasmid followed by Tm (C) or Tg (D) treatment. ChIP-quantitative-PCR assays were carried out to quantify the abundance of XBP-1S located on the promoters of BiP, CHOP, and EDEM genes. * P

    Article Snippet: The antibodies used in this study include: anti-EBV LMP1 (ETU001, KeraFAST), anti-XBP-1 (sc-7160), anti-BiP (sc-1501), anti-PCAF (sc-8999), anti-lamin B (sc-373918, Santa Cruz Biotechnology), anti-acetylated lysine (ab21623), anti-GCN5 (ab71965, Abcam), anti-HA (H3663, Sigma), anti-actin (MAB1501, Millipore), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118S, Cell Signaling).

    Techniques: Over Expression, In Vivo, Expressing, Western Blot, Transfection, Concentration Assay, Negative Control, Quantitative RT-PCR, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    GCN5 competes with PCAF in binding to XBP-1S and inhibits the recruitment of PCAF to the XBP-1S target genes (A) 293T cells were co-transfected PCAF and XBP-1S expression vectors with or without a GCN5 plasmid. IP was performed using an anti-XBP-1S antibody, followed by immunoblotting with anti-PCAF or anti-XBP-1 antibodies. The amounts of PCAF and XBP-1S proteins immunoprecipitated by an anti-XBP-1 antibody were quantified as described under “Materials and Methods.” The XBP-1S protein precipitated in the IP against XBP-1 was used as the input to normalize the amount of PCAF protein detected in the IP. The protein inputs were also analyzed by Western blotting. (B) MCF7 cells were co-transfected with the indicated expression plasmids. ChIP was carried out followed by quantitative PCR to quantify the abundance of PCAF on the BiP, CHOP, and EDEM promoters. Cells only transfected with a PCAF vector were used as a negative control. * P

    Journal: Oncotarget

    Article Title: GCN5 inhibits XBP-1S-mediated transcription by antagonizing PCAF action

    doi:

    Figure Lengend Snippet: GCN5 competes with PCAF in binding to XBP-1S and inhibits the recruitment of PCAF to the XBP-1S target genes (A) 293T cells were co-transfected PCAF and XBP-1S expression vectors with or without a GCN5 plasmid. IP was performed using an anti-XBP-1S antibody, followed by immunoblotting with anti-PCAF or anti-XBP-1 antibodies. The amounts of PCAF and XBP-1S proteins immunoprecipitated by an anti-XBP-1 antibody were quantified as described under “Materials and Methods.” The XBP-1S protein precipitated in the IP against XBP-1 was used as the input to normalize the amount of PCAF protein detected in the IP. The protein inputs were also analyzed by Western blotting. (B) MCF7 cells were co-transfected with the indicated expression plasmids. ChIP was carried out followed by quantitative PCR to quantify the abundance of PCAF on the BiP, CHOP, and EDEM promoters. Cells only transfected with a PCAF vector were used as a negative control. * P

    Article Snippet: The antibodies used in this study include: anti-EBV LMP1 (ETU001, KeraFAST), anti-XBP-1 (sc-7160), anti-BiP (sc-1501), anti-PCAF (sc-8999), anti-lamin B (sc-373918, Santa Cruz Biotechnology), anti-acetylated lysine (ab21623), anti-GCN5 (ab71965, Abcam), anti-HA (H3663, Sigma), anti-actin (MAB1501, Millipore), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118S, Cell Signaling).

    Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Domain study of GCN5 and PCAF (A) Diagram of GCN5 truncations. All the constructs were GFP-tagged. PCAF HD , PCAF homology domain; AT , acetyl transferase domain; Bromo , bromo domain. (B) 293T cells were co-transfected with a XBP-1S and an indicated plasmid to express an individual GCN5 deletion. IP was performed using the anti-XBP-1 antibody followed by Western blotting with anti-GFP or anti-XBP-1 antibodies. Normal IgG (IgG) was used as a negative control. (C) Diagram of PCAF truncations. All the constructs were GFP-tagged. (D) 293T cells were co-transfected with a XBP-1S and an indicated plasmid to express an individual PCAF deletion. IP was performed using the anti-XBP-1 antibody followed by Western blotting with anti-GFP or anti-XBP-1 antibodies.

    Journal: Oncotarget

    Article Title: GCN5 inhibits XBP-1S-mediated transcription by antagonizing PCAF action

    doi:

    Figure Lengend Snippet: Domain study of GCN5 and PCAF (A) Diagram of GCN5 truncations. All the constructs were GFP-tagged. PCAF HD , PCAF homology domain; AT , acetyl transferase domain; Bromo , bromo domain. (B) 293T cells were co-transfected with a XBP-1S and an indicated plasmid to express an individual GCN5 deletion. IP was performed using the anti-XBP-1 antibody followed by Western blotting with anti-GFP or anti-XBP-1 antibodies. Normal IgG (IgG) was used as a negative control. (C) Diagram of PCAF truncations. All the constructs were GFP-tagged. (D) 293T cells were co-transfected with a XBP-1S and an indicated plasmid to express an individual PCAF deletion. IP was performed using the anti-XBP-1 antibody followed by Western blotting with anti-GFP or anti-XBP-1 antibodies.

    Article Snippet: The antibodies used in this study include: anti-EBV LMP1 (ETU001, KeraFAST), anti-XBP-1 (sc-7160), anti-BiP (sc-1501), anti-PCAF (sc-8999), anti-lamin B (sc-373918, Santa Cruz Biotechnology), anti-acetylated lysine (ab21623), anti-GCN5 (ab71965, Abcam), anti-HA (H3663, Sigma), anti-actin (MAB1501, Millipore), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118S, Cell Signaling).

    Techniques: Construct, Transfection, Plasmid Preparation, Western Blot, Negative Control

    XBP-1S interacts with GCN5 through its specific C-terminal region (A) 293T cells were co-transfected with a GCN5 and an indicated XBP-1 expression plasmid (XBP-1U or XBP-1S). IP was performed by incubating the cell lysates prepared from the transfected cells with anti-XBP-1 (A) or anti-GCN5 (B) antibodies. Normal IgG (IgG) was used as a negative control and non-specific protein bands were marked with asterisks. The immunoprecipitated complexes and the protein inputs were analyzed by Western blotting. (C) Nuclear extracts prepared from cells treated with or without Tm were analyzed IP using an anti-XBP-1 antibody. Tm was dissolved in DMSO and the final concentration of DMSO in the culture was kept at 0.1%. Cells treated with 0.1% DMSO were served as a negative control. The immunoprecipitated complexes and the protein inputs were analyzed by Western blotting. (D) Diagram of XBP-1 truncations. All the constructs were HA-tagged. B , basic domain; ZIP , leucine zipper domain. (E) 293T cells were co-transfected with a GCN5 and an indicated plasmid to express an individual XBP-1 deletion. IP was performed using the anti-GCN5 antibody followed by Western blotting with anti-HA or anti-XBP-1 antibodies.

    Journal: Oncotarget

    Article Title: GCN5 inhibits XBP-1S-mediated transcription by antagonizing PCAF action

    doi:

    Figure Lengend Snippet: XBP-1S interacts with GCN5 through its specific C-terminal region (A) 293T cells were co-transfected with a GCN5 and an indicated XBP-1 expression plasmid (XBP-1U or XBP-1S). IP was performed by incubating the cell lysates prepared from the transfected cells with anti-XBP-1 (A) or anti-GCN5 (B) antibodies. Normal IgG (IgG) was used as a negative control and non-specific protein bands were marked with asterisks. The immunoprecipitated complexes and the protein inputs were analyzed by Western blotting. (C) Nuclear extracts prepared from cells treated with or without Tm were analyzed IP using an anti-XBP-1 antibody. Tm was dissolved in DMSO and the final concentration of DMSO in the culture was kept at 0.1%. Cells treated with 0.1% DMSO were served as a negative control. The immunoprecipitated complexes and the protein inputs were analyzed by Western blotting. (D) Diagram of XBP-1 truncations. All the constructs were HA-tagged. B , basic domain; ZIP , leucine zipper domain. (E) 293T cells were co-transfected with a GCN5 and an indicated plasmid to express an individual XBP-1 deletion. IP was performed using the anti-GCN5 antibody followed by Western blotting with anti-HA or anti-XBP-1 antibodies.

    Article Snippet: The antibodies used in this study include: anti-EBV LMP1 (ETU001, KeraFAST), anti-XBP-1 (sc-7160), anti-BiP (sc-1501), anti-PCAF (sc-8999), anti-lamin B (sc-373918, Santa Cruz Biotechnology), anti-acetylated lysine (ab21623), anti-GCN5 (ab71965, Abcam), anti-HA (H3663, Sigma), anti-actin (MAB1501, Millipore), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118S, Cell Signaling).

    Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, Immunoprecipitation, Western Blot, Concentration Assay, Construct

    Txnip regulates Xbp1s in vivo A-D Relative transcript levels of Xbp1s, Erdj3, Serp1, and Edem1 measured by qPCR normalized to 18S from liver samples from wild-type (WT) and Txnip-null (KO) mice ( n = 5), [* P = 0.04 versus WT in (A); * P = 0.02 versus WT in (B); * P = 0.04 versus WT in (C), * P = 0.03 versus WT in (D)]. E Protein levels of Xbp1 and actin measured by Western blot analyses in liver samples of WT and KO mice. F Relative transcript levels of Xbp1s in liver samples of liver-specific Txnip-KO mice (Cre + ) and littermate controls (Cre − ) treated with vehicle, tauroursodeoxycholic acid (TUDCA), or 4-phenylbutyric acid (PBA) for 21 days ( n = 3–4). * P = 0.019, ** P = 0.005 versus Cre + (vehicle only); *** P = 0.001 versus Cre − (vehicle only). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress

    doi: 10.15252/emmm.201302561

    Figure Lengend Snippet: Txnip regulates Xbp1s in vivo A-D Relative transcript levels of Xbp1s, Erdj3, Serp1, and Edem1 measured by qPCR normalized to 18S from liver samples from wild-type (WT) and Txnip-null (KO) mice ( n = 5), [* P = 0.04 versus WT in (A); * P = 0.02 versus WT in (B); * P = 0.04 versus WT in (C), * P = 0.03 versus WT in (D)]. E Protein levels of Xbp1 and actin measured by Western blot analyses in liver samples of WT and KO mice. F Relative transcript levels of Xbp1s in liver samples of liver-specific Txnip-KO mice (Cre + ) and littermate controls (Cre − ) treated with vehicle, tauroursodeoxycholic acid (TUDCA), or 4-phenylbutyric acid (PBA) for 21 days ( n = 3–4). * P = 0.019, ** P = 0.005 versus Cre + (vehicle only); *** P = 0.001 versus Cre − (vehicle only). Source data are available online for this figure.

    Article Snippet: Anti-Txnip antibody for Western blot analysis was from MBL International (JY2), anti-VDUP1 antibody for immunofluorescence was from Invitrogen, anti-PDIA6 antibody was from Abnova (3B4), anti-FLAG antibody was from Sigma-Aldrich (M2), anti-HA antibody was from Covance (16B12), anti-PDI antibody was from Novus Biologicals (RL77), anti-ubiquitin antibody was from Enzo Life Sciences (FK2), anti-Xbp1 antibody was from Santa Cruz, and anti-actin antibody was from Sigma-Aldrich.

    Techniques: In Vivo, Real-time Polymerase Chain Reaction, Mouse Assay, Western Blot

    Txnip regulates Xbp1s in vitro A Relative transcript levels of Xbp1s measured by qPCR normalized to 18S in mouse embryonic fibroblasts (MEFs) from wild-type (WT) and Txnip-null (KO) mice treated with increasing concentrations of tunicamycin for 2 h ( n = 4). 0 μg/ml: *** P = 0.00004 versus WT; 0.5 μg/ml: *** P = 0.00001 versus WT; 1 μg/ml: *** P = 0.0005 versus WT. B Same as in (A) in WT and KO MEFs treated with tunicamycin and chemical chaperones PBA (20 mM) and TUDCA (5 mg/ml) for 6 h ( n = 4). TU/PBA/TUDCA −/−/−: *** P = 0.00008 versus WT; TU/PBA/TUDCA +/−/−: ** P = 0.004 versus WT; TU/PBA/TUDCA +/+/−: *** P = 0.0002 versus WT; TU/PBA/TUDCA +/−/+: *** P = 0.0000003 versus WT. C Same as in (A) in empty vector-transduced (EV) and Txnip-overexpressing (OE) 3T3-L1 fibroblasts treated with tunicamycin for 2 h ( n = 4). 0 μg/ml: ** P = 0.001 versus EV; 1 μg/ml: *** P = 0.00007 versus EV. D Same as in (A) in EV and OE 3T3-L1 fibroblasts transduced with non-targeting (N/T) shRNA, PDI shRNA, or treated with PDI inhibitor 16F16 (5 μM) for 8 h ( n = 4).+/−/−/−: * P = 0.01 versus EV; −/+/−/−: *** P = 0.00008 versus N/T shRNA; −/−/+/−: ** P = 0.002 versus N/T shRNA; −/−/−/+: * P = 0.016 versus N/T shRNA. E-G Protein levels of Xbp1 and actin measured by Western blot analyses in: (E) WT and KO MEFs at increasing durations of treatment with tunicamycin (1 μg/ml), (F) WT and KO MEFs after treatment with tunicamycin (1 μg/ml for 2 h) and chemical chaperones PBA (20 mM) and TUDCA (5 mg/ml), and (G) EV and Txnip 3T3-L1 fibroblasts with and without tunicamycin treatment (1 μg/ml for 2 h). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress

    doi: 10.15252/emmm.201302561

    Figure Lengend Snippet: Txnip regulates Xbp1s in vitro A Relative transcript levels of Xbp1s measured by qPCR normalized to 18S in mouse embryonic fibroblasts (MEFs) from wild-type (WT) and Txnip-null (KO) mice treated with increasing concentrations of tunicamycin for 2 h ( n = 4). 0 μg/ml: *** P = 0.00004 versus WT; 0.5 μg/ml: *** P = 0.00001 versus WT; 1 μg/ml: *** P = 0.0005 versus WT. B Same as in (A) in WT and KO MEFs treated with tunicamycin and chemical chaperones PBA (20 mM) and TUDCA (5 mg/ml) for 6 h ( n = 4). TU/PBA/TUDCA −/−/−: *** P = 0.00008 versus WT; TU/PBA/TUDCA +/−/−: ** P = 0.004 versus WT; TU/PBA/TUDCA +/+/−: *** P = 0.0002 versus WT; TU/PBA/TUDCA +/−/+: *** P = 0.0000003 versus WT. C Same as in (A) in empty vector-transduced (EV) and Txnip-overexpressing (OE) 3T3-L1 fibroblasts treated with tunicamycin for 2 h ( n = 4). 0 μg/ml: ** P = 0.001 versus EV; 1 μg/ml: *** P = 0.00007 versus EV. D Same as in (A) in EV and OE 3T3-L1 fibroblasts transduced with non-targeting (N/T) shRNA, PDI shRNA, or treated with PDI inhibitor 16F16 (5 μM) for 8 h ( n = 4).+/−/−/−: * P = 0.01 versus EV; −/+/−/−: *** P = 0.00008 versus N/T shRNA; −/−/+/−: ** P = 0.002 versus N/T shRNA; −/−/−/+: * P = 0.016 versus N/T shRNA. E-G Protein levels of Xbp1 and actin measured by Western blot analyses in: (E) WT and KO MEFs at increasing durations of treatment with tunicamycin (1 μg/ml), (F) WT and KO MEFs after treatment with tunicamycin (1 μg/ml for 2 h) and chemical chaperones PBA (20 mM) and TUDCA (5 mg/ml), and (G) EV and Txnip 3T3-L1 fibroblasts with and without tunicamycin treatment (1 μg/ml for 2 h). Source data are available online for this figure.

    Article Snippet: Anti-Txnip antibody for Western blot analysis was from MBL International (JY2), anti-VDUP1 antibody for immunofluorescence was from Invitrogen, anti-PDIA6 antibody was from Abnova (3B4), anti-FLAG antibody was from Sigma-Aldrich (M2), anti-HA antibody was from Covance (16B12), anti-PDI antibody was from Novus Biologicals (RL77), anti-ubiquitin antibody was from Enzo Life Sciences (FK2), anti-Xbp1 antibody was from Santa Cruz, and anti-actin antibody was from Sigma-Aldrich.

    Techniques: In Vitro, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation, Transduction, shRNA, Western Blot

    Glutamate stimulation induces local activation of the IRE 1‐ XBP 1 pathway in distal dendrites. Primary cultured mouse hippocampal neurons maintained for 10 days were used for the analyses. (a) Immunofluorescence staining analysis of IRE 1 (green) and MAP 2 (dendritic marker: red). IRE 1 was localized to MAP 2‐positive dendrites (Bar: 20 μm). (b) Western blot analysis of phosphorylated IRE 1 (P‐ IRE 1), IRE 1, phosphorylated eIF 2α (P‐ eIF 2α) and eIF 2α. Cells were treated with 7.5 μM glutamate for the indicated times. P‐ IRE 1 and P‐ eIF 2α were transiently up‐regulated following glutamate stimulation. (c) RT ‐ PCR analysis of Xbp1 and Bip . Spliced Xbp1 ( Xbp1s ) and Bip were transiently induced by treatment with 7.5 μM glutamate for the indicated times ( Xbp1u : unspliced Xbp1 ). (d) Immunofluorescence staining analysis of IRE 1 (green), MAP 2 (blue) and PSD 95 (postsynaptic marker: red). IRE 1 was observed at MAP 2‐positive neurites and PSD 95‐positive postsynaptic sites of dendrites. The localization and level were not significantly changed by the treatment with 7.5 μM glutamate for 1.5 h. The lower panels show high magnification of the dendrites shown in the upper panels. (Bar in upper panels: 20 μm, in lower panels: 5 μm). (e) Immunofluorescence staining analysis of P‐ IRE 1 (green), MAP 2 (blue) and PSD 95 (red). IRE 1 was phosphorylated at PSD 95‐positive postsynaptic sites of dendrites in response to 7.5 μM glutamate exposure for 1.5 h. The lower panels show high magnification of the dendrites shown in the upper panels. Arrow heads indicate immunoreactivities of P‐ IRE 1 overlapped with those of PSD 95. (Bar in upper panels: 20 μm, in lower panels: 5 μm). (f) P‐ IRE 1 fluorescence intensities 40–90 μm from the somata of the dendrites in (e). The fluorescence intensities in these dendrites were not increased by glutamate treatment (mean ± SD , N = 10; number of cells prepared from five independent cultures). (g) The number of P‐ IRE 1‐positive puncta overlapping with PSD 95‐positive postsynaptic sites 40–90 μm from the somata in (e). The number of P‐ IRE 1‐positive postsynaptic sites was increased by glutamate exposure (mean ± SD , N = 10; number of cells prepared from five independent cultures; *** p

    Journal: Journal of Neurochemistry

    Article Title: Neuronal activity‐dependent local activation of dendritic unfolded protein response promotes expression of brain‐derived neurotrophic factor in cell soma

    doi: 10.1111/jnc.14221

    Figure Lengend Snippet: Glutamate stimulation induces local activation of the IRE 1‐ XBP 1 pathway in distal dendrites. Primary cultured mouse hippocampal neurons maintained for 10 days were used for the analyses. (a) Immunofluorescence staining analysis of IRE 1 (green) and MAP 2 (dendritic marker: red). IRE 1 was localized to MAP 2‐positive dendrites (Bar: 20 μm). (b) Western blot analysis of phosphorylated IRE 1 (P‐ IRE 1), IRE 1, phosphorylated eIF 2α (P‐ eIF 2α) and eIF 2α. Cells were treated with 7.5 μM glutamate for the indicated times. P‐ IRE 1 and P‐ eIF 2α were transiently up‐regulated following glutamate stimulation. (c) RT ‐ PCR analysis of Xbp1 and Bip . Spliced Xbp1 ( Xbp1s ) and Bip were transiently induced by treatment with 7.5 μM glutamate for the indicated times ( Xbp1u : unspliced Xbp1 ). (d) Immunofluorescence staining analysis of IRE 1 (green), MAP 2 (blue) and PSD 95 (postsynaptic marker: red). IRE 1 was observed at MAP 2‐positive neurites and PSD 95‐positive postsynaptic sites of dendrites. The localization and level were not significantly changed by the treatment with 7.5 μM glutamate for 1.5 h. The lower panels show high magnification of the dendrites shown in the upper panels. (Bar in upper panels: 20 μm, in lower panels: 5 μm). (e) Immunofluorescence staining analysis of P‐ IRE 1 (green), MAP 2 (blue) and PSD 95 (red). IRE 1 was phosphorylated at PSD 95‐positive postsynaptic sites of dendrites in response to 7.5 μM glutamate exposure for 1.5 h. The lower panels show high magnification of the dendrites shown in the upper panels. Arrow heads indicate immunoreactivities of P‐ IRE 1 overlapped with those of PSD 95. (Bar in upper panels: 20 μm, in lower panels: 5 μm). (f) P‐ IRE 1 fluorescence intensities 40–90 μm from the somata of the dendrites in (e). The fluorescence intensities in these dendrites were not increased by glutamate treatment (mean ± SD , N = 10; number of cells prepared from five independent cultures). (g) The number of P‐ IRE 1‐positive puncta overlapping with PSD 95‐positive postsynaptic sites 40–90 μm from the somata in (e). The number of P‐ IRE 1‐positive postsynaptic sites was increased by glutamate exposure (mean ± SD , N = 10; number of cells prepared from five independent cultures; *** p

    Article Snippet: The following antibodies were used; anti‐IRE1 (1 : 500; Cell Signaling Technology, RRID: AB_823545), anti‐microtubule‐associated protein 2 (MAP2) (1 : 1000; EMD Millipore, Billerica, MA, USA, RRID: AB_11213363), anti‐MAP2 (1 : 500; Abcam, Cambridge, UK, RRID: AB_2138153), anti‐postsynaptic density protein 95 (PSD95) (1 : 500; NeuroMab, Davis, CA, USA, RRID: AB_2307331), anti‐XBP1s (1 : 500; BioLegend, Sandiego, CA, USA, RRID: AB_2562960), and Anti‐phospho‐IRE1α (1 : 500).

    Techniques: Activation Assay, Cell Culture, Immunofluorescence, Staining, Marker, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fluorescence

    Induction of the ER-UPR complex in cells expressing mutant TULP1. (A) Western blot analysis using antibodies against BiP, phosphorylated PERK (pPERK), XBP1 and XBP1s, showed induction of UPR stress proteins in cells expressing mutant forms of TULP1. Actin was used a protein loading control. Western blot experiments were repeated twice. (B) Quantification of ER-stress protein markers was performed using densitometry in mutant TULP1 expressing cells compared to WT-TULP1 expressing cells. The relative band intensity of each protein was normalized to Actin. Data is presented as arbitrary units from two separate western blot densitometry analyses. Statistical significance was calculated using the two-tailed Student’s t -test. * = p

    Journal: PLoS ONE

    Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

    doi: 10.1371/journal.pone.0151806

    Figure Lengend Snippet: Induction of the ER-UPR complex in cells expressing mutant TULP1. (A) Western blot analysis using antibodies against BiP, phosphorylated PERK (pPERK), XBP1 and XBP1s, showed induction of UPR stress proteins in cells expressing mutant forms of TULP1. Actin was used a protein loading control. Western blot experiments were repeated twice. (B) Quantification of ER-stress protein markers was performed using densitometry in mutant TULP1 expressing cells compared to WT-TULP1 expressing cells. The relative band intensity of each protein was normalized to Actin. Data is presented as arbitrary units from two separate western blot densitometry analyses. Statistical significance was calculated using the two-tailed Student’s t -test. * = p

    Article Snippet: Membranes were then probed with primary antibodies against α-actin at 1:10,000 dilution, Tulp1 [ ], BiP, phosphorylated PERK (pPERK), CHOP, XBP1 (which detected both the uncleaved XBP1 and cleaved XBP1s protein fragment), calreticulin, Calnexin and Golgi97 all at 1:1000 dilution (Cell Signaling Technology, Beverly, MA) in antibody buffer (0.2% Triton X-100, 2% BSA, 1X PBS).

    Techniques: Expressing, Mutagenesis, Western Blot, Two Tailed Test

    XBP1 regulates NF-κB via ERα signaling. (A) MCF-7 cells were transfected with XBP1 mutants together with the pGL3-Basic or ERE-luc luciferase construct. At 24 h posttransfection, cells were lysed and luciferase activity was measured. (B)

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: XBP1 regulates NF-κB via ERα signaling. (A) MCF-7 cells were transfected with XBP1 mutants together with the pGL3-Basic or ERE-luc luciferase construct. At 24 h posttransfection, cells were lysed and luciferase activity was measured. (B)

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Transfection, Luciferase, Construct, Activity Assay

    XBP1(S) regulates the RelA protein expression level. (A) Representation of the XBP1 mutant constructs used in this study. (B) Antiestrogen-sensitive breast MCF-7 cells were transfected with various forms of XBP1 mutants together with pGL3-Basic or pGL3-NF-κB-luc

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: XBP1(S) regulates the RelA protein expression level. (A) Representation of the XBP1 mutant constructs used in this study. (B) Antiestrogen-sensitive breast MCF-7 cells were transfected with various forms of XBP1 mutants together with pGL3-Basic or pGL3-NF-κB-luc

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Expressing, Mutagenesis, Construct, Transfection

    p65/RelA inhibition leads to increased apoptosis in XBP1-overexpressing cells. (A) XBP1(S)-, XBP1(U)-ES48-, or LacZ control-overexpressing MCF-7 cells were transfected with control or p65/RelA siRNA for 24 h before being treated with 1 μM 4-OHT

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: p65/RelA inhibition leads to increased apoptosis in XBP1-overexpressing cells. (A) XBP1(S)-, XBP1(U)-ES48-, or LacZ control-overexpressing MCF-7 cells were transfected with control or p65/RelA siRNA for 24 h before being treated with 1 μM 4-OHT

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Inhibition, Transfection

    XBP1 regulates NF-κB signaling by regulating p65/RelA transcription. Three antiestrogen-resistant breast cancer variants (LCC9, MCF-7/RR, LY2) were treated with control siRNA or two different XBP1 siRNAs for 72 h. (A to C) Cells were cotransfected

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: XBP1 regulates NF-κB signaling by regulating p65/RelA transcription. Three antiestrogen-resistant breast cancer variants (LCC9, MCF-7/RR, LY2) were treated with control siRNA or two different XBP1 siRNAs for 72 h. (A to C) Cells were cotransfected

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques:

    XBP1 inhibition sensitizes antiestrogen-resistant breast cancer cells to antiestrogens. (A) LCC9 cells were treated with control (Ctl) siRNA or two different XBP1 siRNAs (XBP1#1 and XBP1#2 siRNAs). At 24 h posttransfection, cells were treated with 100

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: XBP1 inhibition sensitizes antiestrogen-resistant breast cancer cells to antiestrogens. (A) LCC9 cells were treated with control (Ctl) siRNA or two different XBP1 siRNAs (XBP1#1 and XBP1#2 siRNAs). At 24 h posttransfection, cells were treated with 100

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Inhibition, CTL Assay

    XBP1 expression correlates with p65/RelA expression in multiple ERα+ breast cancer data sets.

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: XBP1 expression correlates with p65/RelA expression in multiple ERα+ breast cancer data sets.

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Expressing

    XBP1-overexpressing tumors are resistant to tamoxifen in human breast cancer cell xenografts. (A) XBP1(S)-, XBP1(U)-ES48-, or LacZ control-overexpressing MCF-7 cells (2 × 10 6 ) were injected into the mammary fat pad of athymic nude mice, and tumors

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: XBP1-overexpressing tumors are resistant to tamoxifen in human breast cancer cell xenografts. (A) XBP1(S)-, XBP1(U)-ES48-, or LacZ control-overexpressing MCF-7 cells (2 × 10 6 ) were injected into the mammary fat pad of athymic nude mice, and tumors

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Injection, Mouse Assay

    NF-κB signaling is required for XBP1-mediated antiestrogen resistance. (A) Lysates from MCF-7 cells overexpressing LacZ, XBP1(S), and XBP1(U)-ES48 were subjected to Western blot hybridization with the antibodies indicated. (B) MCF-7 cells overexpressing

    Journal: Molecular and Cellular Biology

    Article Title: NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

    doi: 10.1128/MCB.00847-14

    Figure Lengend Snippet: NF-κB signaling is required for XBP1-mediated antiestrogen resistance. (A) Lysates from MCF-7 cells overexpressing LacZ, XBP1(S), and XBP1(U)-ES48 were subjected to Western blot hybridization with the antibodies indicated. (B) MCF-7 cells overexpressing

    Article Snippet: Depletion of XBP1 strongly inhibited cell proliferation under basal conditions ( ).

    Techniques: Western Blot, Hybridization

    SubAB induces RIDD in B cells. (A–D) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB for 24 h. The mRNA levels of Hspa5 (A), Pdia4 (B), XBP1s (C), and μS (D) were measured by quantitative RT-PCR. (E) 2-d LPS-stimulated WT and S729A B cells were exposed to 5 µg/ml Tu for 24 h. The μS mRNA levels were measured by quantitative RT-PCR. (F–J) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB for 24 h. The mRNA levels of the indicated genes were measured by quantitative RT-PCR. (K and L) Naive B cells from WT and XBP1 KO mice were stimulated with LPS for 3 d. Some XBP1 KO B cells were incubated with 20 µM B-I09 (K) or 5 µM KIRA6 (L) in the last 24 h of LPS stimulation. The μS mRNA levels were measured by quantitative RT-PCR. Data are shown as means ± SD. Quantitative RT-PCR data are representative of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: SubAB induces RIDD in B cells. (A–D) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB for 24 h. The mRNA levels of Hspa5 (A), Pdia4 (B), XBP1s (C), and μS (D) were measured by quantitative RT-PCR. (E) 2-d LPS-stimulated WT and S729A B cells were exposed to 5 µg/ml Tu for 24 h. The μS mRNA levels were measured by quantitative RT-PCR. (F–J) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB for 24 h. The mRNA levels of the indicated genes were measured by quantitative RT-PCR. (K and L) Naive B cells from WT and XBP1 KO mice were stimulated with LPS for 3 d. Some XBP1 KO B cells were incubated with 20 µM B-I09 (K) or 5 µM KIRA6 (L) in the last 24 h of LPS stimulation. The μS mRNA levels were measured by quantitative RT-PCR. Data are shown as means ± SD. Quantitative RT-PCR data are representative of three independent experiments.

    Article Snippet: The following antibodies were obtained commercially: IRE1 (Cell Signaling Technology), XBP1s (Cell Signaling Technology), GRP94 (Stressgen), p97 (Fitzgerald Industries), μ (SouthernBiotech), κ (SouthernBiotech), phospho-ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho–nuclear factor κ–light chain enhancer of activated B cells (NF-κB) p105 (Cell Signaling Technology), phospho–NF-κB p65 (Cell Signaling Technology), and caspase 3 (Cell Signaling Technology).

    Techniques: Quantitative RT-PCR, Mouse Assay, Incubation

    Immunized S729A mice produce increased serum levels of IgM and IgG2b. (A–C) Serum levels of anti–NP IgM in WT, XBP1 KO , S729A, and S729A/XBP1 KO mice ( n = 5 per group) were determined by ELISA on day 3 (A), day 6 (B), and day 9 (C) after immunization with NP-Ficoll on day 0. (D and E) On day 9, NP-Ficoll–immunized mice were sacrificed to analyze for anti–NP IgM–secreting plasma cells in the bone marrow by ELISPOT (D). Bone marrow cells isolated from immunized mice were stained with CD138-PE, B220-BV605, and XBP1s–Alexa Fluor 647. Gated CD138 + populations were analyzed for the expression of XBP1s (E). (F) Serum levels of anti–HEL IgG2b in WT and S729A mice immunized with HEL/CFA followed by 3 boosts with HEL/IFA were determined by ELISA. Data are shown as means ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: Immunized S729A mice produce increased serum levels of IgM and IgG2b. (A–C) Serum levels of anti–NP IgM in WT, XBP1 KO , S729A, and S729A/XBP1 KO mice ( n = 5 per group) were determined by ELISA on day 3 (A), day 6 (B), and day 9 (C) after immunization with NP-Ficoll on day 0. (D and E) On day 9, NP-Ficoll–immunized mice were sacrificed to analyze for anti–NP IgM–secreting plasma cells in the bone marrow by ELISPOT (D). Bone marrow cells isolated from immunized mice were stained with CD138-PE, B220-BV605, and XBP1s–Alexa Fluor 647. Gated CD138 + populations were analyzed for the expression of XBP1s (E). (F) Serum levels of anti–HEL IgG2b in WT and S729A mice immunized with HEL/CFA followed by 3 boosts with HEL/IFA were determined by ELISA. Data are shown as means ± SEM.

    Article Snippet: The following antibodies were obtained commercially: IRE1 (Cell Signaling Technology), XBP1s (Cell Signaling Technology), GRP94 (Stressgen), p97 (Fitzgerald Industries), μ (SouthernBiotech), κ (SouthernBiotech), phospho-ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho–nuclear factor κ–light chain enhancer of activated B cells (NF-κB) p105 (Cell Signaling Technology), phospho–NF-κB p65 (Cell Signaling Technology), and caspase 3 (Cell Signaling Technology).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Isolation, Staining, Expressing, Immunofluorescence

    S729A B cells respond to LPS or CpG-1826 stimulation by producing XBP1s. (A) Naive WT and S729A B cells were stimulated with LPS (20 µg/ml) for a course of 3 d and immunoblotted. (B) Naive WT B cells were stimulated with LPS or CpG-1826 (0.5 µM) for a course of 3 d, stained for XBP1s and B220, and analyzed for gated B220 + /XBP1s + populations. (C) B cells purified from WT and S729A mice were stimulated with LPS for 1 d and continued to be stimulated with LPS for an additional 3, 6, 12, and 24 h (the end of day 2). At each time point, B cells were stained for XBP1s and B220 and were analyzed for gated B220 + /XBP1s + populations. Flow cytofluorometric data are representative of three independent experiments. (D) Naive B cells purified from WT ( n = 3) and S729A ( n = 3) mice were stimulated with LPS for 1 d and continued to be stimulated with LPS for an additional 3, 12, and 24 h. At each time point, B cells were stained with XBP1s–Alexa Fluor 647 and B220–Alexa Fluor 488. The gated B220 + populations were analyzed for expression of XBP1s shown as MFI (means ± SD). The MFI data are representative of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: S729A B cells respond to LPS or CpG-1826 stimulation by producing XBP1s. (A) Naive WT and S729A B cells were stimulated with LPS (20 µg/ml) for a course of 3 d and immunoblotted. (B) Naive WT B cells were stimulated with LPS or CpG-1826 (0.5 µM) for a course of 3 d, stained for XBP1s and B220, and analyzed for gated B220 + /XBP1s + populations. (C) B cells purified from WT and S729A mice were stimulated with LPS for 1 d and continued to be stimulated with LPS for an additional 3, 6, 12, and 24 h (the end of day 2). At each time point, B cells were stained for XBP1s and B220 and were analyzed for gated B220 + /XBP1s + populations. Flow cytofluorometric data are representative of three independent experiments. (D) Naive B cells purified from WT ( n = 3) and S729A ( n = 3) mice were stimulated with LPS for 1 d and continued to be stimulated with LPS for an additional 3, 12, and 24 h. At each time point, B cells were stained with XBP1s–Alexa Fluor 647 and B220–Alexa Fluor 488. The gated B220 + populations were analyzed for expression of XBP1s shown as MFI (means ± SD). The MFI data are representative of three independent experiments.

    Article Snippet: The following antibodies were obtained commercially: IRE1 (Cell Signaling Technology), XBP1s (Cell Signaling Technology), GRP94 (Stressgen), p97 (Fitzgerald Industries), μ (SouthernBiotech), κ (SouthernBiotech), phospho-ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho–nuclear factor κ–light chain enhancer of activated B cells (NF-κB) p105 (Cell Signaling Technology), phospho–NF-κB p65 (Cell Signaling Technology), and caspase 3 (Cell Signaling Technology).

    Techniques: Staining, Purification, Mouse Assay, Flow Cytometry, Expressing

    HEL-immunized S729A mice generate significantly less CD138 + plasma cells in the spleens and bone marrow, and those plasma cells express less XBP1s. (A) 3-d LPS-stimulated WT and S729A B cells were radiolabeled for 4 h and chased in the absence or presence of 1 nM SubAB for a course of 8 h. Lysates were immunoprecipitated for IRE1, which was then analyzed by SDS-PAGE and autoradiography. (B) Human H929 cells were treated with 5 mM DTT for 3 h and immunoblotted. (C) Mouse A20 cells were pretreated with 20 µM B-I09 for 12 h, were subsequently exposed to 1.5 nM SubAB for 12 h, and were immunoblotted. (D) WT and S729A mice were immunized with HEL plus CFA and boosted three times with HEL plus IFA. Bone marrow cells isolated from immunized mice were stained for CD138, B220, and XBP1s. Gated B220 + and CD138 + populations were analyzed for expression of XBP1s. FSC-A, forward scatter area. (E) Percentages of CD138 + plasma cells in the bone marrow of HEL-immunized WT and S729A mice. (F) MFI of XBP1s in CD138 + plasma cells in the bone marrow of HEL-immunized WT and S729A mice. (G) Percentages of CD138 + plasma cells in spleens of HEL-immunized WT and S729A mice. (H) MFI of XBP1s in CD138 + plasma cells in spleens of HEL-immunized WT and S729A mice. (I and J) Serum levels of anti–HEL IgG (I) and IgG1 (J) in immunized WT and S729A mice were determined by ELISA. Data are shown as means ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: HEL-immunized S729A mice generate significantly less CD138 + plasma cells in the spleens and bone marrow, and those plasma cells express less XBP1s. (A) 3-d LPS-stimulated WT and S729A B cells were radiolabeled for 4 h and chased in the absence or presence of 1 nM SubAB for a course of 8 h. Lysates were immunoprecipitated for IRE1, which was then analyzed by SDS-PAGE and autoradiography. (B) Human H929 cells were treated with 5 mM DTT for 3 h and immunoblotted. (C) Mouse A20 cells were pretreated with 20 µM B-I09 for 12 h, were subsequently exposed to 1.5 nM SubAB for 12 h, and were immunoblotted. (D) WT and S729A mice were immunized with HEL plus CFA and boosted three times with HEL plus IFA. Bone marrow cells isolated from immunized mice were stained for CD138, B220, and XBP1s. Gated B220 + and CD138 + populations were analyzed for expression of XBP1s. FSC-A, forward scatter area. (E) Percentages of CD138 + plasma cells in the bone marrow of HEL-immunized WT and S729A mice. (F) MFI of XBP1s in CD138 + plasma cells in the bone marrow of HEL-immunized WT and S729A mice. (G) Percentages of CD138 + plasma cells in spleens of HEL-immunized WT and S729A mice. (H) MFI of XBP1s in CD138 + plasma cells in spleens of HEL-immunized WT and S729A mice. (I and J) Serum levels of anti–HEL IgG (I) and IgG1 (J) in immunized WT and S729A mice were determined by ELISA. Data are shown as means ± SEM.

    Article Snippet: The following antibodies were obtained commercially: IRE1 (Cell Signaling Technology), XBP1s (Cell Signaling Technology), GRP94 (Stressgen), p97 (Fitzgerald Industries), μ (SouthernBiotech), κ (SouthernBiotech), phospho-ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho–nuclear factor κ–light chain enhancer of activated B cells (NF-κB) p105 (Cell Signaling Technology), phospho–NF-κB p65 (Cell Signaling Technology), and caspase 3 (Cell Signaling Technology).

    Techniques: Mouse Assay, Immunoprecipitation, SDS Page, Autoradiography, Immunofluorescence, Isolation, Staining, Expressing, Enzyme-linked Immunosorbent Assay

    A model depicting S729 phosphorylation of IRE1 enhancing the expression of XBP1s and activating RIDD in B cells.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: A model depicting S729 phosphorylation of IRE1 enhancing the expression of XBP1s and activating RIDD in B cells.

    Article Snippet: The following antibodies were obtained commercially: IRE1 (Cell Signaling Technology), XBP1s (Cell Signaling Technology), GRP94 (Stressgen), p97 (Fitzgerald Industries), μ (SouthernBiotech), κ (SouthernBiotech), phospho-ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho–nuclear factor κ–light chain enhancer of activated B cells (NF-κB) p105 (Cell Signaling Technology), phospho–NF-κB p65 (Cell Signaling Technology), and caspase 3 (Cell Signaling Technology).

    Techniques: Expressing

    LPS-stimulated S729A B cells fail to respond to secondary ER stress by producing additional XBP1s. (A) 2-d LPS-stimulated WT and S729A B cells were exposed to 20 µM B-I09 or 5 mM DTT for 3 h, stained for XBP1s and B220, and gated for B220 + /XBP1s + populations. (B) 2-d LPS-stimulated WT and S729A B cells were exposed to 2.5 µM Tg for the indicated times, stained for XBP1s and B220, and gated for B220 + /XBP1s + populations. (C) 2-d LPS-stimulated WT and S729A B cells were exposed to 1.5 nM SubAB for the indicated times, stained for XBP1s and B220, and gated for B220 + /XBP1s + populations. (D) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB, 5 µg/ml Tu, or 2.5 µM Tg for 12 h or were exposed to 5 mM DTT for 3 h and immunoblotted. (E) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB or 2.5 µM Tg for 12 h or were exposed to 5 mM DTT for 3 h, lysed, and immunoprecipitated for IRE1. Bead-bound IRE1 was treated with CIP for 3 h and immunoblotted for IRE1. Data in this figure are representative of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: LPS-stimulated S729A B cells fail to respond to secondary ER stress by producing additional XBP1s. (A) 2-d LPS-stimulated WT and S729A B cells were exposed to 20 µM B-I09 or 5 mM DTT for 3 h, stained for XBP1s and B220, and gated for B220 + /XBP1s + populations. (B) 2-d LPS-stimulated WT and S729A B cells were exposed to 2.5 µM Tg for the indicated times, stained for XBP1s and B220, and gated for B220 + /XBP1s + populations. (C) 2-d LPS-stimulated WT and S729A B cells were exposed to 1.5 nM SubAB for the indicated times, stained for XBP1s and B220, and gated for B220 + /XBP1s + populations. (D) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB, 5 µg/ml Tu, or 2.5 µM Tg for 12 h or were exposed to 5 mM DTT for 3 h and immunoblotted. (E) 2-d LPS-stimulated WT and S729A B cells were exposed to 1 nM SubAB or 2.5 µM Tg for 12 h or were exposed to 5 mM DTT for 3 h, lysed, and immunoprecipitated for IRE1. Bead-bound IRE1 was treated with CIP for 3 h and immunoblotted for IRE1. Data in this figure are representative of three independent experiments.

    Article Snippet: The following antibodies were obtained commercially: IRE1 (Cell Signaling Technology), XBP1s (Cell Signaling Technology), GRP94 (Stressgen), p97 (Fitzgerald Industries), μ (SouthernBiotech), κ (SouthernBiotech), phospho-ERK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho–nuclear factor κ–light chain enhancer of activated B cells (NF-κB) p105 (Cell Signaling Technology), phospho–NF-κB p65 (Cell Signaling Technology), and caspase 3 (Cell Signaling Technology).

    Techniques: Staining, Immunoprecipitation

    CHIKV-induced autophagy is regulated by ER and oxidative stress. (A–C) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of IRE1α (p-IRE1α) and JNK (p-JNK) as well as the formation of spliced form of XBP1 (XBP1s) and the conjugation of LC3 (LC3-II). (A) IRE1α and GAPDH were also followed to control protein expression and loading. Similar results were observed in two independent experiments. (B) Immunofluorescence was performed using anti-pIRE1α and anti-E3 antibody. Bars, 15 µm. (C) The number of cells positive for pIRE1α in the E3 + (infected cells) and E3 − (uninfected cells) populations is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (D and E) WT MEFs were pretreated with control siRNA or siRNA against IRE1α for 3 d followed by infection with CHIKV for 24 h at MOI 1. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (F and G) WT MEFs were incubated for 24 h in control media (ø) or in the presence of CHIKV (MOI = 1). (F) Immunofluorescence was performed using an ROS/RNS detection kit that specifically stains oxygen species and free NO. As positive controls for ROS or RNS induction, WT MEFs were incubated for 5 h with pycocyanin and l -arginine, respectively. Bars, 10 µm. (G) Percentage of cells containing ROS or NO among infected with CHIKV and/or pretreated with specific inhibitor of ROS and RNS as indicated is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in two independent experiments. (H) WT MEFs or cells pretreated with siRNA against IRE1α for 3 d were infected with CHIKV for 24 h in presence of a ROS inhibitor. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (I) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of mTOR (p-mTOR), S6KI (p-S6K1), and AMPK (p-AMPK). mTOR, S6K1, AMPK, and GAPDH were also followed to control protein expression and loading. As control to ROS implication, similar experiments were performed in cells pretreated with ROS inducer and/or ROS inhibitor. Black lines indicate that intervening lanes were spliced out. Similar results were observed in three independent experiments. Student’s test: **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Chikungunya virus-induced autophagy delays caspase-dependent cell death

    doi: 10.1084/jem.20110996

    Figure Lengend Snippet: CHIKV-induced autophagy is regulated by ER and oxidative stress. (A–C) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of IRE1α (p-IRE1α) and JNK (p-JNK) as well as the formation of spliced form of XBP1 (XBP1s) and the conjugation of LC3 (LC3-II). (A) IRE1α and GAPDH were also followed to control protein expression and loading. Similar results were observed in two independent experiments. (B) Immunofluorescence was performed using anti-pIRE1α and anti-E3 antibody. Bars, 15 µm. (C) The number of cells positive for pIRE1α in the E3 + (infected cells) and E3 − (uninfected cells) populations is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (D and E) WT MEFs were pretreated with control siRNA or siRNA against IRE1α for 3 d followed by infection with CHIKV for 24 h at MOI 1. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (F and G) WT MEFs were incubated for 24 h in control media (ø) or in the presence of CHIKV (MOI = 1). (F) Immunofluorescence was performed using an ROS/RNS detection kit that specifically stains oxygen species and free NO. As positive controls for ROS or RNS induction, WT MEFs were incubated for 5 h with pycocyanin and l -arginine, respectively. Bars, 10 µm. (G) Percentage of cells containing ROS or NO among infected with CHIKV and/or pretreated with specific inhibitor of ROS and RNS as indicated is depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in two independent experiments. (H) WT MEFs or cells pretreated with siRNA against IRE1α for 3 d were infected with CHIKV for 24 h in presence of a ROS inhibitor. The number of LC3 punctas per cell and the amount of LC3-II are depicted. Data shown represent mean ± SEM for triplicate samples of > 100 cells per experimental condition. Similar results were observed in three independent experiments. (I) WT MEFs were infected with CHIKV at indicated time points and Western blotting was performed to detect phosphorylation of mTOR (p-mTOR), S6KI (p-S6K1), and AMPK (p-AMPK). mTOR, S6K1, AMPK, and GAPDH were also followed to control protein expression and loading. As control to ROS implication, similar experiments were performed in cells pretreated with ROS inducer and/or ROS inhibitor. Black lines indicate that intervening lanes were spliced out. Similar results were observed in three independent experiments. Student’s test: **, P

    Article Snippet: Proteins were transferred to PVDF membrane and blotted with anti-LC3 (mouse monoclonal; Cell Signaling Technology), anti-Atg5 (mouse monoclonal; Cell Signaling Technology), anti-ATG7 (mouse monoclonal; Cell Signaling Technology), anti-IRE1 (rabbit polyclonal; Abcam), anti-pIRE1 (rabbit polyclonal; Abcam), anti-pJNK (rabbit polyclonal; Abcam), anti-XBP1 (rabbit polyclonal; Abcam), anti-pmTOR (rabbit polyclonal; Abcam), anti-mTOR (rabbit polyclonal; Abcam), anti-pS6K1 (rabbit polyclonal; Abcam), anti-pAMPK (rabbit polyclonal; Abcam), or anti-GAPDH (rabbit polyclonal; Cell Signaling Technology).

    Techniques: Infection, Western Blot, Conjugation Assay, Expressing, Immunofluorescence, Incubation

    miR-30a* inhibits X-box binding protein 1 expression without regulating the levels of activating transcription factor 6 and NF-YA–C in HAPI cells. A and B: qRT-PCR (A) and Western blot analysis (B) of XBP1 expression in HAPI cells transfected with miR-mimics, anti-miR, or corresponding NC oligonucleotides; C and D: qRT-PCR analysis of ATF6 (C) and NF-YA–C (D) expression in HAPI cells after transfection of miR-mimics, anti-miR, or corresponding NC oligonucleotides. a P

    Journal: World Journal of Stem Cells

    Article Title: Bone marrow mesenchymal stem cells induce M2 microglia polarization through PDGF-AA/MANF signaling

    doi: 10.4252/wjsc.v12.i7.633

    Figure Lengend Snippet: miR-30a* inhibits X-box binding protein 1 expression without regulating the levels of activating transcription factor 6 and NF-YA–C in HAPI cells. A and B: qRT-PCR (A) and Western blot analysis (B) of XBP1 expression in HAPI cells transfected with miR-mimics, anti-miR, or corresponding NC oligonucleotides; C and D: qRT-PCR analysis of ATF6 (C) and NF-YA–C (D) expression in HAPI cells after transfection of miR-mimics, anti-miR, or corresponding NC oligonucleotides. a P

    Article Snippet: The primary antibodies were: Polyclonal rabbit anti-MANF antibody (1:500, PA5-20432; Thermo Fisher Scientific); polyclonal rabbit anti-PDGF-AA antibody (1:500, ab216619; Abcam); polyclonal rabbit anti-XBP1 antibody (1:1000, ab37152; Abcam); polyclonal rabbit anti-ATF6 antibody (1:1000, ab203119; Abcam); and monoclonal rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1000, 5174; Cell Signaling Technology, Danvers, MA, United States).

    Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection

    PBA inhibits ER stress and apoptosis in TG-treated endothelial cells. Endothelial cells were treated with TG (10 μM) or together with PBA (1 mM) or PBA (1 mM) alone for 6 h. A. Representative western blots and quantification data of ER stress markers GRP78, PDI in each group cells. B. Representative western blots and quantification data of ER stress markers ATF4 and XBP-1 in each group cells. C. Representative western blots and quantification data of apoptosis-regulating proteins CHOP, Caspase-12, in each group cells. D. MTT results of PBA-treated endothelial cells induced by TG. All experiments were repeated three times. All data represent Mean values ± SEM, * P

    Journal: American Journal of Translational Research

    Article Title: Phenylbutyrate prevents disruption of blood-spinal cord barrier by inhibiting endoplasmic reticulum stress after spinal cord injury

    doi:

    Figure Lengend Snippet: PBA inhibits ER stress and apoptosis in TG-treated endothelial cells. Endothelial cells were treated with TG (10 μM) or together with PBA (1 mM) or PBA (1 mM) alone for 6 h. A. Representative western blots and quantification data of ER stress markers GRP78, PDI in each group cells. B. Representative western blots and quantification data of ER stress markers ATF4 and XBP-1 in each group cells. C. Representative western blots and quantification data of apoptosis-regulating proteins CHOP, Caspase-12, in each group cells. D. MTT results of PBA-treated endothelial cells induced by TG. All experiments were repeated three times. All data represent Mean values ± SEM, * P

    Article Snippet: Anti-XBP-1, β-catenin, P120, Cleaved caspase-12 and PDI were purchased from Abcam.

    Techniques: Western Blot, MTT Assay

    BLOC1S1 degradation and XBP1 splicing are temporally separate. (A and C) Relative expression of BLOC1S1 in NCI-H929 or RPMI-8226 cells treated with the indicated stressors over time. BLOC1S1 mRNA was measured by SYBR green qPCR and normalized to the GAPDH control and is presented relative to time zero. (B and D) Western blots showing phospho-IRE1, IRE1, and β-actin in the cells treated for panel A. Also shown is agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site from the samples used in panel A. (E to L) TaqMan qPCR measurements of BLOC1S1 , XBP1u , and XBP1s for cells treated as indicated. The data were normalized to the GAPDH control and are presented relative to untreated cells. The data are representative of two independent experiments, and error bars show SEM from three technical replicates.

    Journal: Molecular and Cellular Biology

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    doi: 10.1128/MCB.00013-15

    Figure Lengend Snippet: BLOC1S1 degradation and XBP1 splicing are temporally separate. (A and C) Relative expression of BLOC1S1 in NCI-H929 or RPMI-8226 cells treated with the indicated stressors over time. BLOC1S1 mRNA was measured by SYBR green qPCR and normalized to the GAPDH control and is presented relative to time zero. (B and D) Western blots showing phospho-IRE1, IRE1, and β-actin in the cells treated for panel A. Also shown is agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site from the samples used in panel A. (E to L) TaqMan qPCR measurements of BLOC1S1 , XBP1u , and XBP1s for cells treated as indicated. The data were normalized to the GAPDH control and are presented relative to untreated cells. The data are representative of two independent experiments, and error bars show SEM from three technical replicates.

    Article Snippet: The following TaqMan gene expression assays were used (Life Technologies) with TaqMan gene expression master mix (Life Technologies): GAPDH (glyceraldehyde-3-phosphate dehydrogenase), Hs02758991_g1; BLOC1S1 , Hs00155241_m1; FADS2 , Hs00188654_m1; MRC2 , Hs00195862_m1; LRP1 , Hs00233856_m1; ABCA3 , Hs00975530_m1; and XBP1s , Hs03929085_g1; XBP1u, Hs02856596_m1.

    Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Degradation of BLOC1S1 is stressor dependent. (A) Relative quantities of BLOC1S1 in NCI-H929 and RPMI-8226 cells treated with the indicated stressors for 4 h. The mRNA level was measured by SYBR green qPCR and normalized to GAPDH and is shown relative to untreated cells. The data are means ± SEM of three independent experiments. (B) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel A. (C) Quantification of percent XBP1 splicing from the gel images shown in panel B. The data are means ± SEM of three independent experiments. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    doi: 10.1128/MCB.00013-15

    Figure Lengend Snippet: Degradation of BLOC1S1 is stressor dependent. (A) Relative quantities of BLOC1S1 in NCI-H929 and RPMI-8226 cells treated with the indicated stressors for 4 h. The mRNA level was measured by SYBR green qPCR and normalized to GAPDH and is shown relative to untreated cells. The data are means ± SEM of three independent experiments. (B) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel A. (C) Quantification of percent XBP1 splicing from the gel images shown in panel B. The data are means ± SEM of three independent experiments. *, P

    Article Snippet: The following TaqMan gene expression assays were used (Life Technologies) with TaqMan gene expression master mix (Life Technologies): GAPDH (glyceraldehyde-3-phosphate dehydrogenase), Hs02758991_g1; BLOC1S1 , Hs00155241_m1; FADS2 , Hs00188654_m1; MRC2 , Hs00195862_m1; LRP1 , Hs00233856_m1; ABCA3 , Hs00975530_m1; and XBP1s , Hs03929085_g1; XBP1u, Hs02856596_m1.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Inhibition of RIDD does not affect RPMI-8226 cell viability under acute ER stress. (A) Agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in samples from RPMI-8226 cells pretreated for the indicated times with the indicated concentration of 4μ8c and then treated with 2 mM DTT for 90 min. −, untreated cells; DTT, DTT alone. (B) SYBR green qPCR showing relative expression of BLOC1S1 mRNA in RPMI-8226 cells treated with 30 μM 4μ8c at −10 min ( t = −10) (pretreatment) or at 15 min of DTT (post-DTT) and treated with DTT at time zero ( t = 0) for 90 min before washout and recovery for the indicated times. DMSO, vehicle control-treated cells. (C and D) TaqMan qPCR quantification of XBP1u (C) and XBP1s (D) in the samples used for panel B. (E) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples from panel B. The arrows indicate the times of addition of the indicated treatments or washout. (F) SYBR green qPCR of relative CHOP expression in the samples used for panel B. (G) WST-1 cell viability assays of RPMI-8226 cells treated as for panel B or treated with 4μ8c or DMSO without DTT treatment. The data are relative to untreated cells; 0.5% Triton X-100 after DTT washout is shown as a control for maximal cell death. All the graphs show means ± SEM from four (B, F, and G) or three (C and D) independent experiments. n.s., P > 0.05 (unpaired t test). All qPCR measurements were normalized to GAPDH and are presented relative to time −10 for DMSO-treated cell samples.

    Journal: Molecular and Cellular Biology

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    doi: 10.1128/MCB.00013-15

    Figure Lengend Snippet: Inhibition of RIDD does not affect RPMI-8226 cell viability under acute ER stress. (A) Agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in samples from RPMI-8226 cells pretreated for the indicated times with the indicated concentration of 4μ8c and then treated with 2 mM DTT for 90 min. −, untreated cells; DTT, DTT alone. (B) SYBR green qPCR showing relative expression of BLOC1S1 mRNA in RPMI-8226 cells treated with 30 μM 4μ8c at −10 min ( t = −10) (pretreatment) or at 15 min of DTT (post-DTT) and treated with DTT at time zero ( t = 0) for 90 min before washout and recovery for the indicated times. DMSO, vehicle control-treated cells. (C and D) TaqMan qPCR quantification of XBP1u (C) and XBP1s (D) in the samples used for panel B. (E) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples from panel B. The arrows indicate the times of addition of the indicated treatments or washout. (F) SYBR green qPCR of relative CHOP expression in the samples used for panel B. (G) WST-1 cell viability assays of RPMI-8226 cells treated as for panel B or treated with 4μ8c or DMSO without DTT treatment. The data are relative to untreated cells; 0.5% Triton X-100 after DTT washout is shown as a control for maximal cell death. All the graphs show means ± SEM from four (B, F, and G) or three (C and D) independent experiments. n.s., P > 0.05 (unpaired t test). All qPCR measurements were normalized to GAPDH and are presented relative to time −10 for DMSO-treated cell samples.

    Article Snippet: The following TaqMan gene expression assays were used (Life Technologies) with TaqMan gene expression master mix (Life Technologies): GAPDH (glyceraldehyde-3-phosphate dehydrogenase), Hs02758991_g1; BLOC1S1 , Hs00155241_m1; FADS2 , Hs00188654_m1; MRC2 , Hs00195862_m1; LRP1 , Hs00233856_m1; ABCA3 , Hs00975530_m1; and XBP1s , Hs03929085_g1; XBP1u, Hs02856596_m1.

    Techniques: Inhibition, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing

    BLOC1S1 is a RIDD target in myeloma cells. (A) TaqMan qPCR measuring expression of consistently identified RIDD targets containing a consensus IRE1 target sequence in stressed or unstressed myeloma cell lines with or without ActD. (B) Relative BLOC1S1 expression measured by SYBR green qPCR in the indicated myeloma cell lines treated with 2 mM DTT in the presence or absence of DMSO vehicle control or 4μ8c pretreatment. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel B. The qPCR data are normalized to GAPDH mRNA and are expressed relative to untreated cells. (A and B) The data are means ± standard errors of the mean (SEM) from three independent experiments. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    doi: 10.1128/MCB.00013-15

    Figure Lengend Snippet: BLOC1S1 is a RIDD target in myeloma cells. (A) TaqMan qPCR measuring expression of consistently identified RIDD targets containing a consensus IRE1 target sequence in stressed or unstressed myeloma cell lines with or without ActD. (B) Relative BLOC1S1 expression measured by SYBR green qPCR in the indicated myeloma cell lines treated with 2 mM DTT in the presence or absence of DMSO vehicle control or 4μ8c pretreatment. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel B. The qPCR data are normalized to GAPDH mRNA and are expressed relative to untreated cells. (A and B) The data are means ± standard errors of the mean (SEM) from three independent experiments. *, P

    Article Snippet: The following TaqMan gene expression assays were used (Life Technologies) with TaqMan gene expression master mix (Life Technologies): GAPDH (glyceraldehyde-3-phosphate dehydrogenase), Hs02758991_g1; BLOC1S1 , Hs00155241_m1; FADS2 , Hs00188654_m1; MRC2 , Hs00195862_m1; LRP1 , Hs00233856_m1; ABCA3 , Hs00975530_m1; and XBP1s , Hs03929085_g1; XBP1u, Hs02856596_m1.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Sequencing, SYBR Green Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    BLOC1S1 degradation is dispensable for recovery from acute stress. (A) SYBR green qPCR quantification of relative expression of BLOC1S1 in RPMI-8226 cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for the indicated times. (B and C) TaqMan qPCR quantification of XBP1u (B) and XBP1s (C) in the samples used for panel A. (D) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel A. (E) SYBR green qPCR quantification of relative expression of CHOP in the samples used for panel A. (F) Relative cell viability measured by WST-1 assay of cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for 24 h. Triton X-100 (0.5%) treatment after DTT washout in untransduced cells is shown as a control for maximal cell death. The measurements are background-subtracted absorbances relative to the absorbance of unstressed cells for each transduced sample. All qPCR measurements were normalized to GAPDH and are presented relative to 0 min of DTT treatment in DMSO-treated cells. All the graphs show means ± SEM of three (A to C and E) or four (F) independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    doi: 10.1128/MCB.00013-15

    Figure Lengend Snippet: BLOC1S1 degradation is dispensable for recovery from acute stress. (A) SYBR green qPCR quantification of relative expression of BLOC1S1 in RPMI-8226 cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for the indicated times. (B and C) TaqMan qPCR quantification of XBP1u (B) and XBP1s (C) in the samples used for panel A. (D) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel A. (E) SYBR green qPCR quantification of relative expression of CHOP in the samples used for panel A. (F) Relative cell viability measured by WST-1 assay of cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for 24 h. Triton X-100 (0.5%) treatment after DTT washout in untransduced cells is shown as a control for maximal cell death. The measurements are background-subtracted absorbances relative to the absorbance of unstressed cells for each transduced sample. All qPCR measurements were normalized to GAPDH and are presented relative to 0 min of DTT treatment in DMSO-treated cells. All the graphs show means ± SEM of three (A to C and E) or four (F) independent experiments.

    Article Snippet: The following TaqMan gene expression assays were used (Life Technologies) with TaqMan gene expression master mix (Life Technologies): GAPDH (glyceraldehyde-3-phosphate dehydrogenase), Hs02758991_g1; BLOC1S1 , Hs00155241_m1; FADS2 , Hs00188654_m1; MRC2 , Hs00195862_m1; LRP1 , Hs00233856_m1; ABCA3 , Hs00975530_m1; and XBP1s , Hs03929085_g1; XBP1u, Hs02856596_m1.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, WST-1 Assay

    Inhibition of RIDD does not affect NCI-H929 cell viability under acute ER stress. (A) WST-1 cell viability assays of NCI-H929 cells treated as indicated with 2 mM DTT for 60 min (from time zero) and either pretreated (at time −10 min) or posttreated (at time 15 min) with 30 μM 4μ8c and then washed and allowed to recover for 24 h. Cells treated with 0.5% Triton X-100 were included as a dead-cell control. (B) SYBR green qPCR quantification of BLOC1S1 in the DTT-treated samples from panel A. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the DTT-treated samples from panel A. The timing of treatments is indicated below the gel images. (D and E) TaqMan qPCR quantification of XBP1u (D) or XBP1s (E) in the DTT-treated samples from panel A. (F) SYBR green qPCR of relative CHOP expression in the DTT-treated samples from panel A. All qPCR data are normalized to GAPDH and are presented relative to the DMSO control at time −10 min of DTT. All the graphs show means ± SEM from three independent experiments. n.s., P > 0.05 (unpaired t test).

    Journal: Molecular and Cellular Biology

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    doi: 10.1128/MCB.00013-15

    Figure Lengend Snippet: Inhibition of RIDD does not affect NCI-H929 cell viability under acute ER stress. (A) WST-1 cell viability assays of NCI-H929 cells treated as indicated with 2 mM DTT for 60 min (from time zero) and either pretreated (at time −10 min) or posttreated (at time 15 min) with 30 μM 4μ8c and then washed and allowed to recover for 24 h. Cells treated with 0.5% Triton X-100 were included as a dead-cell control. (B) SYBR green qPCR quantification of BLOC1S1 in the DTT-treated samples from panel A. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the DTT-treated samples from panel A. The timing of treatments is indicated below the gel images. (D and E) TaqMan qPCR quantification of XBP1u (D) or XBP1s (E) in the DTT-treated samples from panel A. (F) SYBR green qPCR of relative CHOP expression in the DTT-treated samples from panel A. All qPCR data are normalized to GAPDH and are presented relative to the DMSO control at time −10 min of DTT. All the graphs show means ± SEM from three independent experiments. n.s., P > 0.05 (unpaired t test).

    Article Snippet: The following TaqMan gene expression assays were used (Life Technologies) with TaqMan gene expression master mix (Life Technologies): GAPDH (glyceraldehyde-3-phosphate dehydrogenase), Hs02758991_g1; BLOC1S1 , Hs00155241_m1; FADS2 , Hs00188654_m1; MRC2 , Hs00195862_m1; LRP1 , Hs00233856_m1; ABCA3 , Hs00975530_m1; and XBP1s , Hs03929085_g1; XBP1u, Hs02856596_m1.

    Techniques: Inhibition, SYBR Green Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing

    Plasma Cells from ADAM10 Δ/Δ IgG1-cre +/− mice have altered gene expression. ADAM10 Δ/Δ IgG1-cre +/− and controls were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, splenic PCs were isolated via magnetic bead isolation. mRNA was isolated and (A) Xbp1 (B) Prdm1 , (C) Irf4 and (D) Bcl6 message levels were determined by qPCR. (E) The ratio of Prdm1 to Bcl6 was calculated. Bars represent the mean ± SE of 3 independent studies; cells from 3 mice from each genotype pooled in each study. (*p

    Journal: PLoS ONE

    Article Title: ADAM10 Regulates Transcription Factor Expression Required for Plasma Cell Function

    doi: 10.1371/journal.pone.0042694

    Figure Lengend Snippet: Plasma Cells from ADAM10 Δ/Δ IgG1-cre +/− mice have altered gene expression. ADAM10 Δ/Δ IgG1-cre +/− and controls were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, splenic PCs were isolated via magnetic bead isolation. mRNA was isolated and (A) Xbp1 (B) Prdm1 , (C) Irf4 and (D) Bcl6 message levels were determined by qPCR. (E) The ratio of Prdm1 to Bcl6 was calculated. Bars represent the mean ± SE of 3 independent studies; cells from 3 mice from each genotype pooled in each study. (*p

    Article Snippet: TaqMan gene expression assays included Xbp1 : Mm00457357_m1, Prdm1 : Mm00476128_m1, Bcl6 : Mm00477633_m1; and Irf4 : Mm00516431_m1.

    Techniques: Mouse Assay, Expressing, Isolation, Real-time Polymerase Chain Reaction

    Model. Wild type plasma cells express higher levels of Blimp1, IRF4 and XBP1, while Bcl6 is repressed. This allows for antibody secretion. In the case of ADAM10 Δ/Δ IgG1-cre +/− mice, Bcl6 levels are higher than seen in wild type. Moreover, Blimp1, IRF4 and XBP1 expression are decreased, leading to impaired antibody secretion.

    Journal: PLoS ONE

    Article Title: ADAM10 Regulates Transcription Factor Expression Required for Plasma Cell Function

    doi: 10.1371/journal.pone.0042694

    Figure Lengend Snippet: Model. Wild type plasma cells express higher levels of Blimp1, IRF4 and XBP1, while Bcl6 is repressed. This allows for antibody secretion. In the case of ADAM10 Δ/Δ IgG1-cre +/− mice, Bcl6 levels are higher than seen in wild type. Moreover, Blimp1, IRF4 and XBP1 expression are decreased, leading to impaired antibody secretion.

    Article Snippet: TaqMan gene expression assays included Xbp1 : Mm00457357_m1, Prdm1 : Mm00476128_m1, Bcl6 : Mm00477633_m1; and Irf4 : Mm00516431_m1.

    Techniques: Mouse Assay, Expressing

    XBP1 mRNA is selectively spliced in NBD rats. RNA extracted from CBLM and HC of PND28 control ( n = 5) and NBD ( n = 7) rats was analyzed by RT-PCR using primers that amplify both unspliced XBP1 (XBP1 U ) and spliced (XBP1 S ) XBP1. (A and B) Representative gel images of XBP1 RT-PCR. Note detection of XBP1 U in control rats and both XBP1 U and XBP1 S in NBD rat CBLM (A) or HC (B). (C and D) PstI digestion of XBP1 RT-PCR products. Note PstI-insensitive XBP1 S band in NBD but not control rat CBLM (C) and HC (D). (E) Sequencing of XBP1 U and XBP1 S from CBLM and HC confirms excision of the 26-nucleotide intron from XBP1 S in NBD rats. (F and G) SYBR Green real-time PCR analysis of total XBP1 mRNA in CBLM and HC of control ( n = 5) and NBD ( n = 7) rats. Note that XBP1 mRNA expression levels are unchanged in NBD CBLM (F) but are significantly increased in NBD HC (G: 1.3-fold increase in NBD; ANOVA, P = 0.005). (H and I) Western blot analysis of XBP1 U (33 kDa) protein levels in CBLM and HC cytoplasmic and nuclear extracts from control ( n = 4) and NBD ( n = 4) rats. Representative blots are shown. (H) Relative band densities for CBLM extracts revealed a trend toward decreased XBP1 U protein in cytoplasmic extracts (2.49-fold decrease in NBD; ANOVA, P = 0.065) and a significant decrease in nuclear extracts (1.72-fold decrease in NBD; ANOVA, P = 0.017) from NBD rats compared to controls. (I) Relative band density for HC extracts revealed significant decreases in XBP1 U protein in both cytoplasmic (2.63-fold decrease in NBD; ANOVA, P = 0.033) and nuclear (3.02-fold decrease in NBD; ANOVA, P = 0.018) extracts from NBD rats compared to results for controls. Asterisks indicate P values of

    Journal: Journal of Virology

    Article Title: Endoplasmic Reticulum Stress and Neurodegeneration in Rats Neonatally Infected with Borna Disease Virus

    doi: 10.1128/JVI.00836-06

    Figure Lengend Snippet: XBP1 mRNA is selectively spliced in NBD rats. RNA extracted from CBLM and HC of PND28 control ( n = 5) and NBD ( n = 7) rats was analyzed by RT-PCR using primers that amplify both unspliced XBP1 (XBP1 U ) and spliced (XBP1 S ) XBP1. (A and B) Representative gel images of XBP1 RT-PCR. Note detection of XBP1 U in control rats and both XBP1 U and XBP1 S in NBD rat CBLM (A) or HC (B). (C and D) PstI digestion of XBP1 RT-PCR products. Note PstI-insensitive XBP1 S band in NBD but not control rat CBLM (C) and HC (D). (E) Sequencing of XBP1 U and XBP1 S from CBLM and HC confirms excision of the 26-nucleotide intron from XBP1 S in NBD rats. (F and G) SYBR Green real-time PCR analysis of total XBP1 mRNA in CBLM and HC of control ( n = 5) and NBD ( n = 7) rats. Note that XBP1 mRNA expression levels are unchanged in NBD CBLM (F) but are significantly increased in NBD HC (G: 1.3-fold increase in NBD; ANOVA, P = 0.005). (H and I) Western blot analysis of XBP1 U (33 kDa) protein levels in CBLM and HC cytoplasmic and nuclear extracts from control ( n = 4) and NBD ( n = 4) rats. Representative blots are shown. (H) Relative band densities for CBLM extracts revealed a trend toward decreased XBP1 U protein in cytoplasmic extracts (2.49-fold decrease in NBD; ANOVA, P = 0.065) and a significant decrease in nuclear extracts (1.72-fold decrease in NBD; ANOVA, P = 0.017) from NBD rats compared to controls. (I) Relative band density for HC extracts revealed significant decreases in XBP1 U protein in both cytoplasmic (2.63-fold decrease in NBD; ANOVA, P = 0.033) and nuclear (3.02-fold decrease in NBD; ANOVA, P = 0.018) extracts from NBD rats compared to results for controls. Asterisks indicate P values of

    Article Snippet: Membranes were blocked in 2% nonfat milk powder (for CHOP, ATF4, ATF6, XBP1, and glyceraldehyde phosphate-3-dehydrogenase [GAPDH]) or 2% bovine serum albumin (for phospho-eif2α) in TTBS (20 mM Tris-Hcl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) overnight at room temperature and incubated with mouse monoclonal anti-CHOP (1:100; Santa Cruz Biotechnology), mouse monoclonal anti-PDI (1:500; Affinity BioReagents, Golden, CO), rabbit anti-XBP1 (1:100; M-186; Santa Cruz Biotechnology), mouse monoclonal anti-ATF6 (1:200; IMGENEX, San Diego, CA), or rabbit anti-ATF4 (1:50; C-20; Santa Cruz Biotechnology) in TTBS with 1% nonfat milk or rabbit monoclonal anti-phospho-eif2α (Ser51) (1:300; Cell Signaling Technology, Danvers, MA) in TTBS with 2% bovine serum albumin for 2 h at room temperature.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    Expression of spliced XBP1 and its downstream target genes. qRT-PCR data shows significant differences in transcript levels of spliced XBP1 (A) and ERDJ4 (B) in GADD34 -/- mice compared to WT mice at 72 hours post-SCI.

    Journal: PLoS ONE

    Article Title: Inhibition of GADD34, the Stress-Inducible Regulatory Subunit of the Endoplasmic Reticulum Stress Response, Does Not Enhance Functional Recovery after Spinal Cord Injury

    doi: 10.1371/journal.pone.0109703

    Figure Lengend Snippet: Expression of spliced XBP1 and its downstream target genes. qRT-PCR data shows significant differences in transcript levels of spliced XBP1 (A) and ERDJ4 (B) in GADD34 -/- mice compared to WT mice at 72 hours post-SCI.

    Article Snippet: Target and reference gene PCR amplification was performed in separate tubes with Assay on Demand™ primers (Applied Biosystems) as follows: ATF4 (Mm00515324_m1), CHOP (Mm01135937_g1), Claudin 11 (Mm00500915_m1) GADD34 (Mm00492555_m1), GFAP (Mm00546086_m1), glutamine synthetase (GS: Mm00725701_s1), GRP78 (Mm01333323_g1), microtubule associated protein 2 (Mtap2: Mm00485230_m1), myelin basic protein (MBP: Mm00521980) neuron-specific enolase (NSE: Mm00468052_m1) Olig2 (Mm01210556_m1) and XBP1 (Mm00457359_m1).

    Techniques: Expressing, Quantitative RT-PCR, Mouse Assay

    XBP-1S and XBP-1U interact with Tax. (A) Co-IP was performed with the indicated antibody (i.e., anti-XBP-1 or anti-Tax antibody) and cell lysates (CL) prepared from HEK293 cells transfected with XBP-1S/Tax or XBP-1U/Tax expression vectors. Normal rabbit IgG was used as a negative control. The expression of XBP-1S, XBP-1U, and Tax was confirmed by Western blotting (i.e., CL input). In vitro-translated XBP-1S and XBP-1U (i.e., TNT) were run on the same gel for comparison. The immunoprecipitated complexes were analyzed by Western blotting.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: XBP-1S and XBP-1U interact with Tax. (A) Co-IP was performed with the indicated antibody (i.e., anti-XBP-1 or anti-Tax antibody) and cell lysates (CL) prepared from HEK293 cells transfected with XBP-1S/Tax or XBP-1U/Tax expression vectors. Normal rabbit IgG was used as a negative control. The expression of XBP-1S, XBP-1U, and Tax was confirmed by Western blotting (i.e., CL input). In vitro-translated XBP-1S and XBP-1U (i.e., TNT) were run on the same gel for comparison. The immunoprecipitated complexes were analyzed by Western blotting.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Negative Control, Western Blot, In Vitro, Immunoprecipitation

    XBP-1S and XBP-1U interact with the HTLV-1 LTR in vivo. (A) Schematic representation of the HTLV-1-LTR-F-Luc reporter gene integrated into stable HeLa-HTLV-1-F-luc cells. The locations of the HTLV-1 LTR, the firefly luciferase coding region, the simian virus 40 poly(A) signal, and two primer sets (i.e., HTLV and Luc) for ChIP assays are indicated. The three 21-bp TRE repeats (marked by asterisks) are located within the PCR fragments amplified by the HTLV primer set. (B) Cell lysates prepared from XBP-1U-, XBP-1S-, and CREB1-expressing stable HeLa-HTLV-1-F-luc cells were analyzed by ChIP with anti-XBP-1 antibody (αXBP-1), anti-CREB1 antibody, or control rabbit IgG as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: XBP-1S and XBP-1U interact with the HTLV-1 LTR in vivo. (A) Schematic representation of the HTLV-1-LTR-F-Luc reporter gene integrated into stable HeLa-HTLV-1-F-luc cells. The locations of the HTLV-1 LTR, the firefly luciferase coding region, the simian virus 40 poly(A) signal, and two primer sets (i.e., HTLV and Luc) for ChIP assays are indicated. The three 21-bp TRE repeats (marked by asterisks) are located within the PCR fragments amplified by the HTLV primer set. (B) Cell lysates prepared from XBP-1U-, XBP-1S-, and CREB1-expressing stable HeLa-HTLV-1-F-luc cells were analyzed by ChIP with anti-XBP-1 antibody (αXBP-1), anti-CREB1 antibody, or control rabbit IgG as described in Materials and Methods.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques: In Vivo, Luciferase, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Expressing

    Tax stimulates XBP-1 transcription. HEK293 cells were transiently transfected with the indicated amounts (0.01, 0.05, 0.1, 0.5, and 1 μg) of a Tax-producing plasmid. The empty vector was used as a negative control. Tunicamycin treatment (4 μg/ml) and an ATF6α(1-373) expression vector (1 μg) were used as positive controls. RNAs isolated from the cells were analyzed by quantitative RT-PCR to examine the expression of the genes for XBP-1 and NF-YC.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: Tax stimulates XBP-1 transcription. HEK293 cells were transiently transfected with the indicated amounts (0.01, 0.05, 0.1, 0.5, and 1 μg) of a Tax-producing plasmid. The empty vector was used as a negative control. Tunicamycin treatment (4 μg/ml) and an ATF6α(1-373) expression vector (1 μg) were used as positive controls. RNAs isolated from the cells were analyzed by quantitative RT-PCR to examine the expression of the genes for XBP-1 and NF-YC.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques: Transfection, Plasmid Preparation, Negative Control, Expressing, Isolation, Quantitative RT-PCR

    Effects of mutant Tax proteins on XBP-1 expression. HEK293 cells were transiently transfected with 1 μg of the indicated plasmid to express wild-type Tax (Tax WT), an NF-κB-deficient mutant Tax protein (Tax M22), or a CREB-deficient mutant Tax protein (Tax M47). The empty vector was used as a negative control, while an ATF6α(1-373) expression vector (1 μg) was used as a positive control. RNAs isolated from the cells were analyzed by quantitative RT-PCR to examine the expression of XBP-1.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: Effects of mutant Tax proteins on XBP-1 expression. HEK293 cells were transiently transfected with 1 μg of the indicated plasmid to express wild-type Tax (Tax WT), an NF-κB-deficient mutant Tax protein (Tax M22), or a CREB-deficient mutant Tax protein (Tax M47). The empty vector was used as a negative control, while an ATF6α(1-373) expression vector (1 μg) was used as a positive control. RNAs isolated from the cells were analyzed by quantitative RT-PCR to examine the expression of XBP-1.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques: Mutagenesis, Expressing, Transfection, Plasmid Preparation, Negative Control, Positive Control, Isolation, Quantitative RT-PCR

    Effects of XBP-1 on HTLV-1 LTR-dependent transcription in HEK293, 293T, and HeLa cells. HEK293 (A), 293T (B), and HeLa (C) cells were transiently transfected with HTLV-1-LTR-F-Luc and an expression vector [i.e., ATF6α(1-373), XBP-1S, or XBP-1U]. The empty plasmid (i.e., Mock) was used as a negative control. In a similar set of experiments, HEK293 (D), 293T (E), and HeLa (F) cells were transiently transfected with HTLV-1-LTR-F-Luc with the titration of XBP-1S plasmids (at twofold increments). Relative changes were determined by comparison with the reading of the negative control.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: Effects of XBP-1 on HTLV-1 LTR-dependent transcription in HEK293, 293T, and HeLa cells. HEK293 (A), 293T (B), and HeLa (C) cells were transiently transfected with HTLV-1-LTR-F-Luc and an expression vector [i.e., ATF6α(1-373), XBP-1S, or XBP-1U]. The empty plasmid (i.e., Mock) was used as a negative control. In a similar set of experiments, HEK293 (D), 293T (E), and HeLa (F) cells were transiently transfected with HTLV-1-LTR-F-Luc with the titration of XBP-1S plasmids (at twofold increments). Relative changes were determined by comparison with the reading of the negative control.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, Titration

    Model of cell-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: Model of cell-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques:

    Nuclear colocalization of XBP-1 and Tax. HeLa cells were cotransfected with EGFP-Tax (green) and an XBP-1 expression plasmid. XBP-1U or XBP-1S was immunostained with anti-XBP-1 antibody (red). Colocalization of EGFP-Tax and XBP-1U (or XBP-1S) was detected as yellow color in the overlay image. Nuclei were visualized by DAPI.

    Journal: Journal of Virology

    Article Title: XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription

    doi: 10.1128/JVI.02054-07

    Figure Lengend Snippet: Nuclear colocalization of XBP-1 and Tax. HeLa cells were cotransfected with EGFP-Tax (green) and an XBP-1 expression plasmid. XBP-1U or XBP-1S was immunostained with anti-XBP-1 antibody (red). Colocalization of EGFP-Tax and XBP-1U (or XBP-1S) was detected as yellow color in the overlay image. Nuclei were visualized by DAPI.

    Article Snippet: The samples were then incubated with anti-XBP-1 antibody.

    Techniques: Expressing, Plasmid Preparation

    Effect of nsPEF on the activation of the endoplasmic reticulum (ER) stress sensors IRE1 ( A ) and PERK ( B ). EL-4 cells (top panels) and CT26 cell (bottom panels) were treated with iso-effective doses of 100 and 300 pulses, respectively (200 ns, 7 kV/cm, 10 Hz). Samples were collected at 5 h post treatment. In (A) the expression level of XBP1 in both EL-4 and CT26 was measured by real-time quantitative PCR. The gene mRNA level was normalized to the housekeeping HPRT gene mRNA and is shown as relative expression. In ( B ) phosphorylation of eIF2α was measured by Western blot using an anti-phospho-eIF2α (Serine 51) antibody. Left panels show a representative image for both EL-4 (top panel) and CT26 cells (bottom panel) with eIF2α (phosphorylated and total) and the housekeeping Vinculin protein seen as a 38 and 140 kDa band, respectively. Graphs on the right are the quantifications of the p-eIF2α expressed as fold to sham. 1 µM thaspigargin (Thaps.) was used as a positive control for ER stress induction. Mean +/− s.e. n = 3 for both A and B. * p

    Journal: Cancers

    Article Title: Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer

    doi: 10.3390/cancers11122034

    Figure Lengend Snippet: Effect of nsPEF on the activation of the endoplasmic reticulum (ER) stress sensors IRE1 ( A ) and PERK ( B ). EL-4 cells (top panels) and CT26 cell (bottom panels) were treated with iso-effective doses of 100 and 300 pulses, respectively (200 ns, 7 kV/cm, 10 Hz). Samples were collected at 5 h post treatment. In (A) the expression level of XBP1 in both EL-4 and CT26 was measured by real-time quantitative PCR. The gene mRNA level was normalized to the housekeeping HPRT gene mRNA and is shown as relative expression. In ( B ) phosphorylation of eIF2α was measured by Western blot using an anti-phospho-eIF2α (Serine 51) antibody. Left panels show a representative image for both EL-4 (top panel) and CT26 cells (bottom panel) with eIF2α (phosphorylated and total) and the housekeeping Vinculin protein seen as a 38 and 140 kDa band, respectively. Graphs on the right are the quantifications of the p-eIF2α expressed as fold to sham. 1 µM thaspigargin (Thaps.) was used as a positive control for ER stress induction. Mean +/− s.e. n = 3 for both A and B. * p

    Article Snippet: Gene expression analysis was conducted using TaqMan Gene Expression Assays for spliced XBP1 (Mm03464496_m1) [ ] and the endogenous housekeeping gene HPRT (Mm03024075_m1).

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Positive Control

    Sodium demethylcantharidate induced ER stress in HHC cells. ER stress markers, including p-IRE1, GRP78/BiP, XBP1s, Caspase 12 and CHOP were analyzed in concentration course manner by Western blotting.

    Journal: American Journal of Translational Research

    Article Title: Sodium demethylcantharidate induces apoptosis in hepatocellular carcinoma cells via ER stress

    doi:

    Figure Lengend Snippet: Sodium demethylcantharidate induced ER stress in HHC cells. ER stress markers, including p-IRE1, GRP78/BiP, XBP1s, Caspase 12 and CHOP were analyzed in concentration course manner by Western blotting.

    Article Snippet: Western blot analysis indicated that p-IRE1, GRP78/BiP, XBP1s, caspase-12, and CHOP levels were higher in dissected tumors of the xenograft model exposed to sodium demethylcantharidate ( ).

    Techniques: Concentration Assay, Western Blot

    Knockdown of ERα inhibits ICI-induced UPR signaling. LCC1 and -9 cells were transfected with ERα shRNA or control pcDNA and treated with or without 100 nM ICI for 72 h. A–C ) Western blot hybridization of treated protein homogenates was used to measure GRP78 ( A ), XBP1-S ( B ), and CHOP ( C ) expression. D ) XBP1 and ERE activity in LCC9 cells was determined by using a CRE-luc system ( n = 3). * P

    Journal: The FASEB Journal

    Article Title: Knockdown of estrogen receptor-α induces autophagy and inhibits antiestrogen-mediated unfolded protein response activation, promoting ROS-induced breast cancer cell death

    doi: 10.1096/fj.13-247353

    Figure Lengend Snippet: Knockdown of ERα inhibits ICI-induced UPR signaling. LCC1 and -9 cells were transfected with ERα shRNA or control pcDNA and treated with or without 100 nM ICI for 72 h. A–C ) Western blot hybridization of treated protein homogenates was used to measure GRP78 ( A ), XBP1-S ( B ), and CHOP ( C ) expression. D ) XBP1 and ERE activity in LCC9 cells was determined by using a CRE-luc system ( n = 3). * P

    Article Snippet: Activation of UPR signaling occurred ∼144 h after treatment initiation in LCC1 breast cancer cells and after ∼72 h in the ICI-resistant LCC9 cells, as observed by increased expression of GRP78, XBP1-S, ATF4, cleaved ATF6, and CHOP.

    Techniques: Transfection, shRNA, Western Blot, Hybridization, Expressing, Activity Assay

    ICI differentially activates UPR signaling in antiestrogen-sensitive LCC1 and antiestrogen-resistant LCC9 cells. Representative Western blot images of PERK, phospho-eIF2α, eIF2α, ATF4, CHOP, IRE1α, XBP1-S, cleaved-ATF6, GRP78, GRP94, and β-tubulin (loading control) in LCC1 ( A ) and LCC9 ( B ) breast cancer cells treated with 100 nM ICI for 6 d. Biological replicate Western blots ( n = 3) for each time course were quantified with ImageJ, and data are reported as relative protein expression.

    Journal: The FASEB Journal

    Article Title: Knockdown of estrogen receptor-α induces autophagy and inhibits antiestrogen-mediated unfolded protein response activation, promoting ROS-induced breast cancer cell death

    doi: 10.1096/fj.13-247353

    Figure Lengend Snippet: ICI differentially activates UPR signaling in antiestrogen-sensitive LCC1 and antiestrogen-resistant LCC9 cells. Representative Western blot images of PERK, phospho-eIF2α, eIF2α, ATF4, CHOP, IRE1α, XBP1-S, cleaved-ATF6, GRP78, GRP94, and β-tubulin (loading control) in LCC1 ( A ) and LCC9 ( B ) breast cancer cells treated with 100 nM ICI for 6 d. Biological replicate Western blots ( n = 3) for each time course were quantified with ImageJ, and data are reported as relative protein expression.

    Article Snippet: Activation of UPR signaling occurred ∼144 h after treatment initiation in LCC1 breast cancer cells and after ∼72 h in the ICI-resistant LCC9 cells, as observed by increased expression of GRP78, XBP1-S, ATF4, cleaved ATF6, and CHOP.

    Techniques: Western Blot, Expressing