Journal: The Journal of biological chemistry

Article Title: Activation of the canonical ER stress IRE1-XBP1 pathway by insulin regulates glucose and lipid metabolism.

doi: 10.1016/j.jbc.2022.102283

Figure Lengend Snippet: Figure 3. Insulin activates IRE1-XBP1s signaling in cultured hepatocytes. A, mice were subjected to a 24-h fast, then refed for 1.5 h, blood glucose levels (n = 5) and plasma insulin levels (n = 6 7). B–E, Hepa1-6 cells were cultured in 4- or 25-mM glucose for 1 h, then treated with 10 nM insulin for 2 h. The protein levels of XBP1 and phosphorylation levels of mediators in the insulin signaling pathway and MEK-ERK-p38 signaling were determined (B). Densitometric analysis of the protein levels of XBP1s and XBP1u (C), phosphorylation levels of AKT, GSK, and IRE1 (D), and MEK, ERK, and p38 (E) (n = 3). F, primary hepatocytes were subjected to 3 h serum starvation, then treated with 10 nM of insulin for the indicated time. G, Hepa1-6 cells were subjected to 3-h serum starvation, then treated with 10 nM of insulin for the indicated time. *p < 0.05, Student’s t test. IRE1, inositol-requiring enzyme; XBP1, X-box binding protein 1; XBP1u, XBP1 from an unspliced form; XBP1s, XBP1 from a spliced form.

Article Snippet: For the phosphorylation of IRE1 by AKT1, 0.4 μg of human IRE1 (OriGene) was added to the reaction containing 25 mM Tris–HCl (pH7.5), 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, and 0.1 μg of AKT1 (abcam) with or without 0.2 mM ATP.

Techniques: Cell Culture, Clinical Proteomics, Phospho-proteomics, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Activation of the canonical ER stress IRE1-XBP1 pathway by insulin regulates glucose and lipid metabolism.

doi: 10.1016/j.jbc.2022.102283

Figure Lengend Snippet: Figure 4. Insulin activates IRE1-XBP1s signaling in the liver of fasted mice. A–D, after 12 h of fasting, mice were treated with vehicle or 1 unit/kg of insulin for 15 or 30 min, then liver tissues were collected. The protein levels of XBP1 and phosphorylation levels of mediators in the insulin signaling pathway, canonical ER stress pathway, and MEK-ERK-p38 signaling were determined in immunoblots (A). Densitometric analysis of the phosphorylation levels of AKT, GSK, IRE1, and protein levels of XBP1s (B), phosphorylation levels of MEK, ERK, and p38 (C), phosphorylation levels of pPERK and eIF2a and ATF6 protein levels (D) (n = 3 5). E, the mRNA levels of XBP1s and XBP1u in the liver. Densitometric analysis of the mRNA levels of XBP1s and XBP1u (n = 3 5). Each lane represents an individual mouse sample (A, E). F and G, forty-eight hours after the addition of adenoviral expression vectors of XBP1s or XBP1u, Hepa1-6 cells were treated with cycloheximide (CHX; 50 μg/ml) and harvested at the indicated time point. *p < 0.05, Student’s t test. ER, endo- plasmic reticulum; IRE1, inositol-requiring enzyme; XBP1, X-box binding protein 1; XBP1u, XBP1 from an unspliced form; XBP1s, XBP1 from a spliced form; ER, endoplasmic reticulum.

Article Snippet: For the phosphorylation of IRE1 by AKT1, 0.4 μg of human IRE1 (OriGene) was added to the reaction containing 25 mM Tris–HCl (pH7.5), 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, and 0.1 μg of AKT1 (abcam) with or without 0.2 mM ATP.

Techniques: Phospho-proteomics, Western Blot, Expressing, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Activation of the canonical ER stress IRE1-XBP1 pathway by insulin regulates glucose and lipid metabolism.

doi: 10.1016/j.jbc.2022.102283

Figure Lengend Snippet: Figure 5. AKT directly phosphorylates IRE1. A, Hepa1-6 cells were cultured in Dulbecco’s modified Eagle’s medium without serum for 1 h, then 50 μM of LY294002 or 10 μM of AKT inhibitor II were added. After 1 h of incubation, 10 nM of insulin was added and cells were harvested 3 h later. Each lane represents an individual sample. B, 0.4 μg of IRE1 was incubated with 0.1 μg of AKT1 for 1 h at 37 C in the presence or absence of ATP. The phosphorylation levels of IRE1 at S724 was examined using anti-IRE1S724 phosphorylation-specific antibody. C, AKT1/2 inhibitor was incubated with 0.4 μg of IRE1 for 10 min, then 0.1 μg of AKT1 was added and incubated for 1 h at 37 C. The phosphorylation levels of IRE1 at S724 were examined as in (B). IRE1, inositol- requiring enzyme.

Article Snippet: For the phosphorylation of IRE1 by AKT1, 0.4 μg of human IRE1 (OriGene) was added to the reaction containing 25 mM Tris–HCl (pH7.5), 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, and 0.1 μg of AKT1 (abcam) with or without 0.2 mM ATP.

Techniques: Cell Culture, Incubation, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Activation of the canonical ER stress IRE1-XBP1 pathway by insulin regulates glucose and lipid metabolism.

doi: 10.1016/j.jbc.2022.102283

Figure Lengend Snippet: Figure 7. XBP1u stimulates gluconeogenesis in cultured primary hepatocytes and liver. A–D, liver tissues of C57BL6 mice were collected at fed and fasted (24 h) states. Immunoblots (A), densitometric analysis of the phosphorylation levels of IRE1 (B), and protein levels of XBP1s (C) and XBP1u (D) (n = 5). E, XBP1u augmented PKA-stimulated reporter activity in Hepa1-6 cells cotransfected with 100 ng of XBP1u and XBP1s plasmids and 30 ng of CRE-Luciferase reporter plasmid (n = 3). F, Hepa1-6 cells were cotransfected with 150 ng of pFR-Luciferase reporter plasmid, 20 ng of pFA-CREB, 100 ng of XBP1u, XBP1s, and PKA plasmids (n = 3). G–I, twenty-four hours after the seeding of primary hepatocytes, adenoviral expression vectors of GFP and XBP1u were added. Forty-eight hours later, primary hepatocytes were subjected to 3 h serum starvation, followed by washing with PBS, and addition of glucose production medium and/or 0.2 mM cAMP for 3 h (G) (n = 3). One set of hepatocytes were harvested in TRIzol reagent for the determination of mRNA levels of G6pc (H) and Pck1(I) (n = 3). J, primary hepatocytes were treated with adenoviral shRNA of XBP1 together with adenoviral expression vectors of GFP or XBP1u. Cells were treated as in (G) (n = 3). K–P, three-month-old WT mice were injected (jugular vein) with AAV8-XBP1shRNA (shXBP1-3) (4 × 10

Article Snippet: For the phosphorylation of IRE1 by AKT1, 0.4 μg of human IRE1 (OriGene) was added to the reaction containing 25 mM Tris–HCl (pH7.5), 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, and 0.1 μg of AKT1 (abcam) with or without 0.2 mM ATP.

Techniques: Cell Culture, Western Blot, Phospho-proteomics, Activity Assay, Luciferase, Plasmid Preparation, Expressing, shRNA, Injection

Journal: Frontiers in Neuroscience

Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

doi: 10.3389/fnins.2022.906651

Figure Lengend Snippet: METH and HIV-1 upregulate ER/UPR signaling mediators. Astrocytes were treated with (C,E) METH (50 nM) for 7 days (blue bars) or (D,F) pseudotyped HIV-1 (500 RT) for 7 days (red bars) before (A–D) calcium imaging or (E,F) protein analysis via Simple Wes. (A–D) Astrocytes were transfected with a GFP-calmodulin calcium sensor (GCaMP6s) for 48 h prior to calcium flux analysis. (A,B) Time series confocal imaging was used to measure changes in fluorescence every 500 ms for a total of 5 min (600 cycles). (B) Representative calcium flux line tracings illustrate the change in astrocyte calcium flux (ΔF) at any given time point, with control media or METH (250 μM) at 20 cycles (10 s) and ionomycin (10 μM) at 450 cycles (225 s). Calcium flux was calculated by: ΔF = (F-F 0 )/(F max -F 0 ), where F is the fluorescence intensity at any given time; F 0 is the baseline (1 – 20 cycles) fluorescence intensity, and F max is the maximum fluorescence intensity when exposed to ionomycin (450 – 600 cycles). (C,D) Area under the curve (AUC) was calculated by the sum of ΔF following treatment with control media or METH (250 μM) at 20 cycles (10 sec) and before ionomycin (10 μM) at 450 cycles (225 s). Individual dots represent the average AUC from a minimum of 20 cells per biological donor and are graphed as fold changes. One-way ANOVA was performed for statistical analysis followed by Fisher’s LSD test for stand-alone comparisons to account for sensitivity of calcium flux variation across different biological donors. (E–H) Protein expression of BiP, ATF6, PERK, and IRE1α was measured via Simple Wes post-treatment of (E,G) chronic (7 days) METH (50 nM) or (F,H) HIV-1 infection (7 days; 500 RT). (E,F) Representative blot images are illustrated from two separate biological donors per post-treatment paradigm. (G,H) Data from a minimum of four donors are compiled for graphical representation. Individual dots on graphs represent fold changes to vinculin for separate biological donors. Statistics were performed using ratio-paired t -tests for individual targets per condition.

Article Snippet: The same protocol was used for IRE1α overexpression (cat # SC309043, OriGene, Rockville, MD, United States) and the backbone plasmid (cat # PCMV6XL5, OriGene).

Techniques: Imaging, Transfection, Fluorescence, Control, Expressing, Infection

Journal: Frontiers in Neuroscience

Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

doi: 10.3389/fnins.2022.906651

Figure Lengend Snippet: Inhibition of IRE1α decreases astrocyte metabolic function. (A–K) Astrocytes were treated with pharmacological inhibitors for the three UPR arms [ATF6 (AEBSF; 100 μM), PERK (GSK2606414; 1 μM), IRE1α (STF-083010; 60 μM)] for 3 h prior to (A–J) Seahorse Mito Stress Test for metabolic assessment or (K) extracellular lactate dehydrogenase (LDH) assay for cytotoxicity. Representative (A) OCR and (B) ECAR profile tracings from a single astrocyte donor were graphed over time. Compiled data from five separate biological donors quantifying fold changes in (C) basal respiration, (D) ATP production, (E) maximal respiration, (F) spare respiratory capacity, (G) non-mitochondrial OCR, (H) proton leak, (I) basal ECAR, and (J) maximal ECAR were graphed for statistical comparisons. Statistical significance was determined via one-way ANOVA followed by Tukey’s post hoc for multiple comparisons. Each dot on graphs represents the averaged data from a minimum of six replicates per biological donor.

Article Snippet: The same protocol was used for IRE1α overexpression (cat # SC309043, OriGene, Rockville, MD, United States) and the backbone plasmid (cat # PCMV6XL5, OriGene).

Techniques: Inhibition, Lactate Dehydrogenase Assay

Journal: Frontiers in Neuroscience

Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

doi: 10.3389/fnins.2022.906651

Figure Lengend Snippet: IRE1α inhibition can partially restore astrocyte metabolic function following HIV-1 infection and chronic METH exposure. (A–E) Astrocytes were treated with METH (50 nM; blue bars) or infected with pseudotyped HIV-1 (500 RT; red bars) for 7 days followed by IRE1α pharmacological inhibition (STF-083010, 60 μM) for 3 h prior to Seahorse Mito Stress Test. (A) Representative metabolic OCR profile tracing is illustrated from a single astrocyte donor. Compiled data quantifying fold changes in (B) basal respiration, (C) ATP production, (D) maximal respiration, and (E) spare respiratory capacity were graphed for statistical comparisons. Rescue experiments were performed twice in two separate biological donors to acquire four separate data sets. A minimum of six replicates were performed per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc for multiple comparisons.

Article Snippet: The same protocol was used for IRE1α overexpression (cat # SC309043, OriGene, Rockville, MD, United States) and the backbone plasmid (cat # PCMV6XL5, OriGene).

Techniques: Inhibition, Infection

Journal: Frontiers in Neuroscience

Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

doi: 10.3389/fnins.2022.906651

Figure Lengend Snippet: Astrocyte IRE1α regulates both mitochondrial respiration and glycolytic activity through distinct mechanisms. (A–I) Astrocytes were transfected with an IRE1α overexpression vector (gray bars) or backbone (white bars) and then treated with IL-1β for 24 h (checkered bars) prior to functional assessments. (B,C) Cellular lysates were collected and assayed by Simple Wes to quantify (B) IRE1α and (C) BiP expression levels. Vinculin was used as an internal control. (D,F,G) Mitochondrial respiration and (E,H,I) glycolytic activity were assessed by Seahorse metabolic assay. (D) OCR and (E) ECAR profile tracings from a representative astrocyte donor were graphed over time. Fold changes in (F) basal respiration, (G) maximal respiration, (H) basal ECAR, and (I) maximal ECAR were graphed for statistical comparisons. Individual dots on graphs represent the averaged data from a minimum of 6 replicates per biological donor. Significance was determined by one-way ANOVA and Tukey’s post hoc for multiple comparisons. Experimental Illustration was made with BioRender.com .

Article Snippet: The same protocol was used for IRE1α overexpression (cat # SC309043, OriGene, Rockville, MD, United States) and the backbone plasmid (cat # PCMV6XL5, OriGene).

Techniques: Activity Assay, Transfection, Over Expression, Plasmid Preparation, Functional Assay, Expressing, Control, Metabolic Assay

Journal: Frontiers in Neuroscience

Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

doi: 10.3389/fnins.2022.906651

Figure Lengend Snippet: IRE1α overexpression augments cytokine expression and increases glutamate clearance in human astrocytes. Backbone and IRE1α transfected astrocytes were treated with IL-1β for 24 h. (A–D) Cells were immunolabeled with antibodies specific for IRE1α (red) and the astrocyte marker glial fibrillary acidic protein (GFAP, green). Nuclear DNA was labeled with DAPI (blue). (E–G) Experiments in four separate biological donors were analyzed to quantify morphological activation. Individual dots on graphs represent compiled fold-changes calculated from duplicate wells and/or triplicate images per condition for each biological astrocyte donor. (E) GFAP intensity was measured across full-well scans using SoftMax Pro and normalized to DAPI. (F) Process length of individual astrocytes was manually traced and measured using ImageJ Software. (G) Percent morphological activation was calculated based on the number of astrocytes presenting with ‘reactive’ morphology divided by the total number of astrocytes imaged. (H) CCL2 and (I) CXCL8 levels were assessed by an ELISA, and expression was normalized to metabolic activity prior to calculating fold changes to backbone. (J) Astrocytes were treated with 400 nM glutamate for 24 h. Remaining glutamate levels were quantified by fluorescent assay to calculate% glutamate clearance followed by fold change for each individual donor. Individual dots on graphs represent compiled data from triplicate experiments per biological astrocyte donor. Significance was determined by one-way ANOVA and Tukey’s post hoc for multiple comparisons.

Article Snippet: The same protocol was used for IRE1α overexpression (cat # SC309043, OriGene, Rockville, MD, United States) and the backbone plasmid (cat # PCMV6XL5, OriGene).

Techniques: Over Expression, Expressing, Transfection, Immunolabeling, Marker, Labeling, Activation Assay, Software, Enzyme-linked Immunosorbent Assay, Activity Assay, Fluorescence

Journal: Frontiers in Neuroscience

Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

doi: 10.3389/fnins.2022.906651

Figure Lengend Snippet: Astrocyte IRE1α regulates ER stress, mitochondrial respiration, glycolysis, inflammation, and glutamate clearance.

Article Snippet: The same protocol was used for IRE1α overexpression (cat # SC309043, OriGene, Rockville, MD, United States) and the backbone plasmid (cat # PCMV6XL5, OriGene).

Techniques: Activity Assay

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: Sequences of primer pairs for qPCR

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques:

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: (a) Immunocytochemical staining results in U87 glioblastoma cells (20× magnification): the increased immunoreactivity in 4-HC group is remarkable; (b) Mean optical density values of immunostaining results of control and 4-HC-treated groups in U87 cell line (*p<0.05); (c) Relative gene expression results by qPCR analysis (*p<0.05). Data represent mean ± SD. 4-HC: 4-Hydroperoxy cyclophosphamide-treated group; ATF6: Activating transcription factor 6; eIF2α: Eukaryotic translation initiation factor 2α; GRP78: Glucose-regulated protein 78; mRNA: Messenger ribonucleic acid; p-IRE1α: Phospho-inositol-requiring enzyme 1α; p-PERK: Phospho-protein kinase R (PKR)-like endoplasmic reticulum kinase; qPCR: Quantitative polymerase chain reaction; SD: Standard deviation

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques: Staining, Immunostaining, Control, Gene Expression, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: Numerical data of optical density values for U87 cells

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques:

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: Relative mRNA fold changes for U87 cells

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques:

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: (a) Immunocytochemical staining results in T98 glioblastoma cells (20× magnification): the increased immunoreactivity in 4-HC group is remarkable; (b) Mean optical density values of immunostaining results of control and 4-HC-treated groups in T98 cell line (*p<0.05); (c) Relative gene expression results by qPCR analysis (*p<0.05). Data represent mean ± SD. 4-HC: 4-Hydroperoxy cyclophosphamide-treated group; ATF6: Activating transcription factor 6; eIF2α: Eukaryotic translation initiation factor 2α; GRP78: Glucose-regulated protein 78; mRNA: Messenger ribonucleic acid; p-IRE1α: Phospho-inositol-requiring enzyme 1α; p-PERK: Phospho-protein kinase R (PKR)-like endoplasmic reticulum kinase; qPCR: Quantitative polymerase chain reaction; SD: Standard deviation

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques: Staining, Immunostaining, Control, Gene Expression, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: Numerical data of optical density values for T98 cells

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques:

Journal: Romanian Journal of Morphology and Embryology

Article Title: Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines

doi: 10.47162/RJME.65.1.04

Figure Lengend Snippet: Relative mRNA fold changes for T98 cells

Article Snippet: Thereafter, the blocking solution was removed without washing and phospho (p)-PERK (Cat# bs-3330R, Bioss, USA), ATF6 (Cat# bs-1634R, Bioss), GRP78 (Cat# bs-1219R, Bioss), p-IRE1α (Cat# orb157704, Biorbyt, USA), eukaryotic translation initiation factor 2 alpha (eIF2α) (Cat# bs-4842, Bioss), and caspase-3 (Cat# bs-0081R, Bioss) the primary antibodies were added, and cells were incubated overnight (16±18 hours) at 4°C.

Techniques:

Journal: Stem Cells International

Article Title: Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1 α Branch of the Endoplasmic Reticulum Stress Response

doi: 10.1155/2023/4483776

Figure Lengend Snippet: MSCs attenuated EMT via the IRE1 α /XBP1 pathway. (a) The protein expression levels of IRE1 α and p-IRE1 α were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). (b) The mRNA expression level of IRE1 α was measured using Q-PCR ( n = 3, one-way ANOVA with Duncan's post hoc test). (c) A549 cells were treated with 4 μ 8c (5 and 10 μ M) for 48 hr, and the protein expression levels of IRE1 α and XBP-1s were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). (d) A549 cells were treated with 10 ng/ml TGF- β 1 in the presence or absence of 4 μ 8c for 72 hr. The protein expression levels of p-IRE1 α , XBP-1s, E-cadherin and vimentin were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). The data are shown as the means ± SEMs ( ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05 vs. the control group; ### P < 0.001, ## P < 0.01, # P < 0.05 vs. the TGF- β 1 group).

Article Snippet: After the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to membranes, which were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1 α (A00683-1, Boster, Wuhan, China), phospho-IRE1 α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1 α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET1610-39, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight.

Techniques: Expressing, Western Blot, Software, Control

Journal: Stem Cells International

Article Title: Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1 α Branch of the Endoplasmic Reticulum Stress Response

doi: 10.1155/2023/4483776

Figure Lengend Snippet: MSCs attenuated ER stress and EMT in the lungs of mice with lung fibrosis. (a) The mRNA expression levels of E-cadherin and Vimentin in lung tissues were measured using Q-PCR ( n = 5, one-way ANOVA with Duncan's post hoc test). (b) The protein expression levels of E-cadherin and vimentin in lung tissues were measured using western blotting, and the results were quantified via densitometry by using ImageJ software ( n = 3, one-way ANOVA with Duncan's post hoc test). (c) The mRNA expression levels of Bip , Atf6 , Atf4 , Xbp-1s , Ire1α and Chop in lung tissues were measured using Q-PCR ( n = 5, one-way ANOVA with Duncan's post hoc test). (d) The protein expression levels of ATF6, ATF4, IRE1 α , p-IRE1 α , XBP-1s, XBP-1u, BiP and CHOP in lung tissues were measured using western blotting, and the results were quantified via densitometry by using ImageJ software ( n = 3, one-way ANOVA with Duncan's post hoc test). (e) Images of immunofluorescence staining of BiP (green) and vimentin (red) in the lung tissues of mice. Scale bar, 100 μ m. The data are shown as the means ± SEMs ( ∗∗ P < 0.01, ∗ P < 0.05 vs. the control group; # P < 0.05, ## P < 0.01 vs. the BLM group).

Article Snippet: After the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to membranes, which were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1 α (A00683-1, Boster, Wuhan, China), phospho-IRE1 α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1 α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET1610-39, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight.

Techniques: Expressing, Western Blot, Software, Immunofluorescence, Staining, Control