Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Heatmap showing DEGs specific to ER stress. ( B ) qPCR validation of DEGs specific to ER stress revealed by RNA-seq. ( C ) Immunohistochemical GRP94 expression in peri-infarct myocardium. ( D ) Mice were subjected to MI surgery as described in Methods. GRP94 levels were detected by immunoblot (mean ± SD, n = 4). ( E ) GRP94 attenuates infarct sizes 1 day after MI. TTC staining on the left shows infarct size modulation by AAV2/9-GRP94 overexpression (Ad-GRP94) or siRNA knockdown (Si-GRP94) at 24 hours after MI. Right panel shows the percentage of infarct sizes. n = 4. ( F and G ) GRP94 markedly improves cardiac function after MI. Transthoracic echocardiographic analysis was performed at 1 week after surgery. n = 6. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. * P < 0.05; ** P < 0.01 by unpaired, 2-tailed Student’s t test ( D ) or 1-way ANOVA with Tukey-Kramer post hoc analysis ( E – G ).

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Biomarker Discovery, RNA Sequencing, Immunohistochemical staining, Expressing, Western Blot, Staining, Over Expression, Knockdown

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Cardiomyocytes were exposed to the indicated dose of H 2 O 2 . After 24 hours, cells were harvested for the analysis of GRP94 levels by immunoblot. n = 4. ( B ) Cardiomyocytes were cultivated under hypoxia for the indicated time. After 24 hours, cells were harvested for the analysis of GRP94 levels by immunoblot ( n = 4). Data analyzed versus the untreated group. ( C ) GRP94 attenuates cells’ sensitivity to hypoxia-induced apoptosis. Cardiomyocytes were infected with adenoviruses overexpressing GRP94 (Ad-GRP94) or Glb1 (Ad-Glb1) at MOIs of 50, or transfected with 60 nM GRP94 siRNA (Si-GRP94) or siRNA scramble (Si-NC). After 36 hours, cells were cultivated for another 12 hours under hypoxic conditions. The TUNEL assay was utilized to detect apoptosis. Quantitative analysis of apoptosis is shown in right panel. CON, control. Scale bar: 50 μm. ( D ) GRP94 attenuates mitochondrial fission caused by hypoxia stress. Cardiomyocytes were treated as in C . Representative photos show mitochondrial fission (left). The cells were stained with MitoTracker, and then the ratios of cells with lamentous network, intermediate, or completely fragmented mitochondrial structures were calculated. At least 50 cells were counted from different optical fields of each sample ( n = 4). CON, control. Scale bars: 20 μm. ( E and F ) GRP94 attenuates the cleavage of Opa1 into (S)-Opa1 caused by hypoxia stress. Cardiomyocytes were treated as in C . The levels of MFN1, MFN2, Opa1, DRP1, p-DRP1, and FIS1 were analyzed by immunoblot. n = 4. ( G ) GRP94 attenuated AMI-induced mitochondrial over-fragmentation. Adult male C57BL/6 mice (8–10 weeks old) were injected with AAV2/9 carrying GRP94 , Glb1 , GRP94 siRNA, or siRNA scramble and subjected to permanent left anterior descending coronary artery ligation. The left panels are representative photos of TEM images of cardiac slices. Scale bars: 1 μm (left images) and 100 nm (enlarged images). * P < 0.05; ** P < 0.01 by 1-way ANOVA with Tukey-Kramer post hoc analysis ( A – C , E , and F ) or 2-way ANOVA followed by Bonferroni’s multiple-comparison test ( D ).

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Western Blot, Infection, Transfection, TUNEL Assay, Control, Staining, Injection, Ligation, Comparison

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Hypoxia-induced GRP94 mRNA upregulation is suppressed by Yy1 knockdown. Mouse cardiomyocytes transfected with 40 nM siRNAs targeting Cebpb , c-Jun , Cebpa , Yy1 , or scramble control (Si-NC) were exposed to hypoxia for 12 hours. GRP94 mRNA levels were quantified by qRT-PCR ( n = 4). ( B ) Yy1 knockdown inhibits hypoxia-induced GRP94 protein expression. Cardiomyocytes transfected with Si-Yy1 or Si-NC were subjected to hypoxia. GRP94 protein was analyzed by immunoblot with total protein as loading control ( n = 4). ( C ) Yy1 binds to the GRP94 promoter under hypoxia. ChIP assays were performed using Yy1 or β-actin (negative control) antibodies in cardiomyocytes exposed to hypoxia for the indicated durations. ( D and E ) Yy1 regulates GRP94 protein in a hypoxia-dependent manner. ( D ) GRP94 levels were unaffected by Yy1 overexpression (Ad-Yy1, MOI = 40) under normoxia. ( E ) Ad-Yy1 enhanced hypoxia-induced GRP94 expression ( n = 4). ( F – H ) Yy1 activates GRP94 promoter via direct binding. ( F ) Luciferase reporter assays in MCM cells transfected with GRP94 promoter (pGL-GRP94) or empty vector (pGL-4.17), followed by Ad- Yy1 /Ad- Glb1 (MOI = 40) and hypoxia ( n = 4). ( G ) The –500 to +1 bp promoter region mediates Yy1 responsiveness. ( H ) Mutation of Yy1-binding site (pGL-GRP94-YS-mut) abolishes Yy1-driven activation. ( I ) ER stress response element (ERMS) mutation (pGL-GRP94-ERMS-mut) attenuates Yy1-dependent promoter activity ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group (or vs. Si-NC in A ) by 1-way ANOVA with Tukey-Kramer post hoc analysis ( A , B , and D – G ) or 2-way ANOVA with Bonferroni’s multiple-comparison test ( H and I ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Knockdown, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Negative Control, Over Expression, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Activation Assay, Activity Assay, Comparison

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Heatmap shows differentially expressed lncRNAs in the mouse model after MI. n = 3. ( B ) Hypoxia enhances Yy1-dependent enrichment of Lnc- AI662270 . Cardiomyocytes infected with Ad-Glb1 or Ad-Yy1 (MOI = 40) for 24 hours were exposed to hypoxia (12 hours). RNA immunoprecipitation (RIP) using anti-Yy1 or IgG antibody, followed by qRT-PCR ( n = 3). ( C ) Hypoxia redirects Yy1 binding from Lnc-Oip5os1 to Lnc- AI662270 . RNA pull-down with biotinylated probes (Bio- AI662270 , Bio-Oip5os1, or Bio-NC) in cardiomyocytes. Yy1 binding was analyzed by immunoblot (actin as loading control). ( D ) Subcellular localization of Lnc- AI662270 (cytoplasmic) and Lnc-Oip5os1 (nuclear) under hypoxia. Fluorescence in situ hybridization (FISH) in cardiomyocytes after 12-hour hypoxia. Scale bar: 20 μm. ( E ) Lnc- AI662270 regulates hypoxia-induced GRP94 mRNA. Cardiomyocytes infected with Ad- AI662270 or Ad-Glb1 (MOI = 40), or transfected with 40 nM ASO- AI662270 /ASO-NC for 36 hours, were exposed to hypoxia (12 hours). GRP94 mRNA levels analyzed by qRT-PCR ( n = 4). ( F and G ) AI662270 or Oip5os1 does not alter nuclear Yy1 abundance. ( F ) Nuclear Yy1 levels in cells infected with Ad-Oip5os1 or Ad-Glb1 (MOI = 40). ( G ) Nuclear Yy1 in cells transfected with ASO- AI662270 /ASO-NC. Cells were exposed to hypoxia (12 hours) prior to nuclear fractionation ( n = 4). ( H and I ) Lnc- AI662270 modulates Yy1 occupancy on the GRP94 promoter. ( H ) ASO- AI662270 reduces Yy1 binding to the GRP94 promoter. ( I ) Ad- AI662270 enhances Yy1-promoter interaction. ChIP-qPCR with anti-Yy1 or IgG after 36 hr treatment ( n = 4). ( J and K ) Hypoxia-dependent GRP94 regulation by Lnc- AI662270 . ( J ) ASO- AI662270 attenuates hypoxia-induced GRP94 protein. ( K ) Ad- AI662270 elevates GRP94 under hypoxia. Immunoblots quantified from 48 hours after transfection ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group by 1-way ANOVA with Tukey-Kramer post hoc analysis ( B , E , J , and K ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Infection, RNA Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Western Blot, Control, Fluorescence, In Situ Hybridization, Transfection, Fractionation, ChIP-qPCR

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Hypoxia treatment induces an increase in AI662270 levels. Cardiomyocytes were exposed to hypoxia for the indicated durations. AI662270 mRNA analyzed by qRT-PCR ( n = 4). ( B ) Tunicamycin (TN, 15 mg/L) induces AI662270 expression. Time-course analysis after TN treatment ( n = 4). ( C ) ER stress inhibitor TUDCA (120 mg/L) blocks hypoxia-induced AI662270 upregulation. Cells were pretreated with TUDCA for 2 hours before 12-hour hypoxia ( n = 4). ( D ) AI662270 knockdown specifically attenuates TN-induced GRP94. Cardiomyocytes were transfected with 40 nM ASO- AI662270 /ASO-NC for 24 hours, and then treated with TN (15 mg/L, 12 hours). ER stress markers (IRE1α, GRP78, PDI, GRP94, CHOP) analyzed by immunoblotting ( n = 4). ( E ) AI662270 silencing exacerbates hypoxia-induced Opa1 cleavage. Cells transfected with ASO- AI662270 /ASO-NC were exposed to hypoxia (12 hours). Opa1 protein was analyzed by immunoblot. ( F ) AI662270 deficiency promotes mitochondrial fragmentation. Mitochondrial morphology was assessed by MitoTracker staining (≥100 cells/group). Categories: filamentous (>80% tubular), intermediate (30%–80%), fragmented (<30%). Scale bar: 20 μm ( n = 4). ( G ) AI662270 knockdown enhances hypoxia-induced apoptosis. TUNEL assay in cells treated as in E . Scale bar: 50 μm ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group by 1-way ANOVA with Tukey-Kramer post hoc analysis ( A – E and G ) or 2-way ANOVA with Bonferroni’s multiple-comparison test ( F ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Transfection, Western Blot, Staining, TUNEL Assay, Comparison

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A and B ) AI662270 regulates GRP94 and L-Opa1 in vivo. C57BL/6 mice (8–10 weeks old) injected with AAV2/9- AI662270 (1 × 10 11 ), Ad-Glb1, ASO- AI662270 (40 nM), or ASO-NC underwent permanent LAD ligation. ( A ) GRP94 and Opa1-L protein levels at 24 hours after MI. ( B ) Quantification of L-Opa1/S-Opa1 ratio ( n = 4). ( C and D ) AI662270 suppresses MI-induced mitochondrial fragmentation. ( C ) TEM images of cardiomyocyte mitochondria. ( D ) Quantification of fragmented mitochondria ( n = 3). Scale bar: 1 μm. ( E ansd F ) AI662270 reduces infarct size. ( E ) TTC-stained heart sections (red, viable; white, infarct). ( F ) Infarct area quantification at 24 hours after MI ( n = 4). ( G and H ) AI662270 preserves cardiac function. Echocardiography at 1 week after MI. ( G ) LVEF and ( H ) LVFS ( n = 4). LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. * P < 0.05; ** P < 0.01 versus the indicated group by 1-way ANOVA with Tukey-Kramer post hoc analysis ( B , D , and F – H ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: In Vivo, Injection, Ligation, Staining

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A and B ) Hypoxia and ER stress remodel GRP94 interactomes. Cardiomyocytes exposed to ( A ) hypoxia (12 hours) or ( B ) tunicamycin (TN, 15 mg/L, 12 hours). GRP94 coimmunoprecipitated proteins identified by LC-MS/MS. ( C ) Anxa2 knockdown (si-Anxa2, 40 nM) blocks GRP94-mediated L-Opa1 stabilization. Cells infected with Ad- GRP94 or Ad- Glb1 (MOI = 50) and transfected with si-Anxa2/si-NC for 36 hours, followed by hypoxia (12 hours). Opa1 isoforms analyzed by immunoblotting ( n = 4). ( D ) Endogenous GRP94-Anxa2 interaction under hypoxia. Co-IP with anti-GRP94 or IgG in hypoxic cardiomyocytes (12 hours). ( E ) GRP94 overexpression enhances Anxa2 and p-Akt1 levels. Ad-GRP94–infected cells (MOI = 50, 48 hours). Anxa2, p-Akt1 (Ser473), and L-Opa1 analyzed ( n = 4). ( F ) Anxa2 deletion abrogates GRP94-induced p-Akt1 and L-Opa1. Ad-GRP94 (MOI = 50) + si-Anxa2/si-NC (40 nM, 48 hours). Protein levels quantified ( n = 4). ( G ) Akt1 inhibitor LY294002 (50 μM) reverses GRP94 effects. Ad-GRP94–infected cells treated with LY294002 for 48 hours ( n = 4). Anxa2, p-Akt1, and Opa1 protein was analyzed by immunoblot ( n = 4). ( H ) Anxa2 knockdown negates GRP94-mediated mitochondrial protection. Mitochondrial morphology was assessed by MitoTracker (≥50 cells/group). Categories: filamentous (>80% tubular), intermediate (30%–80%), fragmented (<30%). Scale bar: 20 μm ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group by 2-way ANOVA with Bonferroni’s multiple-comparison test. ( I ) Anxa2 deletion abolishes the GRP94 antiapoptotic effect. TUNEL assay in cells treated as in F . Scale bar: 50 μm ( n = 4). All data are expressed as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Liquid Chromatography with Mass Spectroscopy, Knockdown, Infection, Transfection, Western Blot, Co-Immunoprecipitation Assay, Over Expression, Comparison, TUNEL Assay

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: The red arrow represents the effect of the AI662270 /GRP94 signaling axis activation induced by early hypoxic stress.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Activation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice *

doi: 10.1074/mcp.M116.065474

Figure Lengend Snippet: Validation of the differentially expressed proteins that were identified by MALDI-TOF/TOF-MS. Seven proteins (HSP90B1, beta-actin, albumin, APOA1, CALR, FABP5, and PRDX6) were randomly selected from the differently expressed proteins that were identified by MALDI-TOF/TOF-MS and were validated in transgenic and nontransgenic males (A) and females (B) by Western blot assay. Wt: the liver tissue of wild-type mice; P: the peritumor tissue of transgenic mice; T: the tumor tissue of transgenic mice; Non-Tg, C57BL/6J wild-type nontransgenic mice; Tg: transgenic mice. The numbers indicate different individuals. Bradford reagent was used as the loading control.

Article Snippet: Primary and secondary antibodies: anti-phoshpo-MEK-1/2 polyclonal antibody (diluted 1:1000, Elabscience, Wuhan, China), anti-phoshpo-ERK1/2 monoclonal antibody (diluted 1:2000, Cell Signaling Technology, MA), anti-phoshpo-PI3 Kinase polyclonal antibody (diluted 1:1000, Cell Signaling Technology), anti-phoshpo-Akt monoclonal antibody (diluted 1:2000, Cell Signaling Technology), anti-phoshpo-mTOR monoclonal antibody (diluted 1:1000, Cell Signaling Technology), anti-APOA1 monoclonal antibody (diluted 1:1000, Proteintech, Wuhan, China), anti-β-actin polyclonal antibody (diluted 1:5000, Bioworld Technology Co., Ltd., MN), anti-HSP90B1 polyclonal antibody (diluted 1:1000, Elabscience), anti-albumin polyclonal antibody (diluted 1:1000, Abclonal, Boston, MA), anti-calreticulin polyclonal antibody (diluted 1:1000, Abways, Shanghai, China), anti-fatty acid binding protein 5 polyclonal antibody (diluted 1:500, Proteintech), anti-peroxiredoxin VI monoclonal antibody (diluted 1:2000, Abfrontier, Santiago, CA), anti-mouse-IgG antibody (diluted 1:5000, Bioworld) and anti-rabbit-IgG antibody (diluted 1:5000, Bioworld).

Techniques: Biomarker Discovery, Transgenic Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of Phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in Acquired Middle Ear Cholesteatoma Growth

doi: 10.3390/ijms140714439

Figure Lengend Snippet: 2D side-by-side comparison of the 2-DE Western blot images of HSP27, PRDX2, GRP78 and GRP75 in cholesteatoma tissues and retroauricular skin run with pI 4–7 and pI 3–10, respectively. Images A , B , C and D are for HSP27. Images E , F , G and H are for PRDX2. Images I , J , K and L are for GRP78. Images M , N , O and P are for GRP75.

Article Snippet: Rabbit anti-human HSP27, GRP75, GRP78, GRP94 and PRDX2 antibodies were purchased from ProteinTech Group (Chicago, IL, USA).

Techniques: Comparison, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of Phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in Acquired Middle Ear Cholesteatoma Growth

doi: 10.3390/ijms140714439

Figure Lengend Snippet: Validation of HSP27, GRP75, GRP78, GRP94 and PRDX2 by Western blotting analysis and RT-PCR. The tissues of cholesteatoma and retroauricular skin were collected from six individual patients. Cholesteatoma is presented by C, and retroauricular skin is presented by S. β-actin was used for normalization.

Article Snippet: Rabbit anti-human HSP27, GRP75, GRP78, GRP94 and PRDX2 antibodies were purchased from ProteinTech Group (Chicago, IL, USA).

Techniques: Biomarker Discovery, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: Available sera from 39 patients were sampled 7 days before onset (D-7), the day of acute GvHD onset, and 7 days and 14 days after diagnosis (D+7 and D+14, respectively). Gp96 was quantified by ELISA. (A) All acute GvHD patients included. It is worth noting that, for some patients, data at one specific time point may be missing. Statistical analysis performed using a 1-way ANOVA test (Dunn’s multiple comparisons test). *P < 0.05. (B) Patients (n = 17) with grade II (n = 10) vs. III–IV intestinal (GI) GvHD (n = 7). Statistical analysis performed using a 2-way ANOVA test (Tukey’s multiple comparisons test). **P < 0.01, ****P < 0.0001. (C) Patients presenting isolated skin GvHD (n = 18). (D) Non-GvHD patients (n = 7). Gp96 levels were measured 3 weeks after hematopoietic stem cell transplantation (median: D21, most often the onset of GvHD) and 1 week later (median: D28).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Isolation, Transplantation Assay

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Left panel: mouse model of acute GvHD. Syn: syngeneic graft (no GvHD developed). BM: allogeneic graft without splenocytes (no GvHD). Allo: allogeneic graft with splenocytes (acute GvHD). Right panel: survival of the mice belonging to the different groups. (B) Western blot analysis of Gp96 and HSP90 in sera at day 7 after transplantation. Recombinant proteins were run as a control. (C) Western blot analysis of Gp96 expression at the indicated days after transplantation in sera of mice developing GvHD (Allo) or not (Syn). The lanes were run on the same gel but were noncontiguous. (D) Proportion of splenic macrophages (according to F4/80 staining) expressing Gp96 at the surface was determined by FACS analysis 7 days after hematopoietic stem cell transplantation. Statistical analysis performed using a 1-way ANOVA test. ****P < 0.0001. A representative experiment is shown (n = 3).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Transplantation Assay, Recombinant, Control, Expressing, Staining

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Western blot analysis of Gp96. (B) Coomassie blue coloration of proteins coimmunoprecipitated with Gp96 (bottom panel) in mice developing GvHD (Allo) or not (Syn) 7 days after hematopoietic stem cell transplantation. Ctl, mice with no transplantation. The proteins indicated were determined by mass spectrometry. One representative experiment is shown (n = 2). (C) Protein-protein interaction data between Gp96 or HSP60 immobilized onto the biosensor and C3 (left panel) or C3b (right panel) as analytes.

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Transplantation Assay, Mass Spectrometry

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Linear structure of C3 (β-chain in yellow, α-chain in blue) and of the tested complement C3 fragments (see also Supplemental Figure 2). (B) Yeast two-hybrid assay between the C3 fragments described in A and full-length Gp96. A representative picture is shown. (C) Linear structure of Gp96 and the analyzed deletion mutants. ABD, ATP-binding domain; Ca2+, charged linker domain; PBD, peptide-binding domain. (D) Two-hybrid assay between complement C3 749–1,303 amino acid fragment and Gp96 deletion mutants. A representative image is shown (n = 3).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Y2H Assay, Binding Assay, Two Hybrid Assay

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: The N-terminus of Gp96 is indicated. Each structure is represented by a transparent surface superposed to the ribbon diagram of the protein backbone. The color code is the following: red for the C3 fragment (749–955), blue for the Gp96 and pink for the C3 complementary structure (1–748; 956–1641). (A and B) Two views of the Gp96-C3 fragment model. (C and D) Two views of the Gp96 full-length C3 model. This figure was prepared with PyMOL (http://www.pymol.org). N = N-terminal domain.

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques:

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Gp96 inhibits C3b cleavage by factors I and H. Upper panel: Western blot analysis of the α’-chain (C3b) and the α2-chain (iC3b and C3c) both labeled with anti-C3c antibody, and of the α1-dg-chain (iC3b) labeled with anti-C3d antibody, after C3b incubation (0.3 μM) with normal human serum, supplemented with factors I and H with or without purified human Gp96 (1.2 μM). Lower panel: densitometry quantification. Cleavage without Gp96 is rationalized to 1. Statistical analysis performed using a 2-tailed Mann-Whitney U test. *P < 0.05; **P < 0.01 (n = 4 or 5). (B) Gp96 effect on opsonophagocytosis. Flow cytometry analysis of phagocytosis by human purified macrophages of Alexa Fluor 488–conjugated (AF 488–conjugated) E. coli bioparticles after opsonization by healthy human serum with or without Gp96 (1.5 mM). A representative image of AF 488 fluorescence in living cells is shown: the filled gray curve represents opsonization without serum, gray line with serum alone, black line with serum and Gp96, and dotted black line with serum and “BSA in buffer.” Histograms of mean fluorescence intensity ± SEM are shown. Statistical analysis performed using a one-tailed Mann-Whitney U test. *P < 0.05 (n = 3). (C) Gp96 effect on opsonization. AF 488–E. coli bioparticles were incubated with serum with or without Gp96 (1.5 mM). C3 and C4 were determined by Western blot in supernatants after deesterification of proteins tagged on bioparticles. One representative experiment is shown (n = 3). (D) Microscopy on AF 488–bioparticles after C3 AF568 staining. Right panel: C3 bioparticle quantification (median of fluorescence of AF 568 staining ± SEM). Statistical analysis performed using a one-tailed Mann-Whitney U test. *P < 0.05 (n = 3). (E and F) Gp96 effect on complement activation pathways. (E) Antibody-coated sheep erythrocytes (for classical pathway) (n = 6) and (F) rabbit erythrocytes hemolysis (for alternative pathway) (n = 4), in presence of Gp96 or controls. Percent of hemolysis are shown. Statistical analysis performed using a 2-tailed Mann-Whitney U test. *P < 0.05; **P < 0.01 (E: n = 6; F: n = 4).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Labeling, Incubation, Purification, MANN-WHITNEY, Flow Cytometry, Fluorescence, One-tailed Test, Microscopy, Staining, Activation Assay

Journal: JCI Insight

Article Title: Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

doi: 10.1172/jci.insight.90531

Figure Lengend Snippet: (A) Left, schematic representation of the model of GvHD used with a C3–/– recipient: FvB/N → WT or C3–/– C57BL/6. Right, Western blot analysis of C3 and Gp96 in day 7 sera of WT or C3–/– mice, developing GvHD (Allo) or not (Syn and BM). (B) Top, schematic representation of the mice model with a C3–/– donor: WT or C3–/– C57BL/6 → BALB/c. Lower left panel, Western blot analysis of C3 and Gp96 in day 7 sera of mice receiving a WT or a C3–/– graft, developing GvHD (Allo) or not (Syn and BM). Samples from 7 animals per group were pooled. Lower right panel, immunoprecipitation of Gp96 in the sera from the different described animals groups was followed by complement C3 immunoblotting. One representative experiment out of 3 performed is shown. (C) Survival of the mice belonging to the different groups described in B, (n = 4) from 2 independent experiments. (D) Percentage of activated, splenic T cells (CD3+CD69+) 7 days after hematopoietic stem cell transplantation in mice receiving either a WT or a C3–/– graft, developing GvHD (Allo) or not (Syn and BM). Statistical analysis performed using a one-tailed Mann-Whitney U test. *P < 0.05 (n = 3).

Article Snippet: Healthy volunteer sera were incubated at 37°C for 1 hour with Gp96 produced in the human cell line HEK293 (BioVendor).

Techniques: Western Blot, Immunoprecipitation, Transplantation Assay, One-tailed Test, MANN-WHITNEY