Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: HSP90B1 and HSPA5 genes and GP96 are induced in ALD. (A) mRNA abundance of HSP90B1 and HSPA5 genes (in transcripts per million) was evaluated by RNA sequencing in patients with normal liver (n = 10), early ASH (n = 12), severe_AH (n = 17), explants_AH (n = 10), and livers from NAFLD (n = 10), HCV (n = 10), and comp_Cirrhosis (n = 9). (B) Representative immunohistochemistry of GP96 in human normal and alcoholic liver (magnification × 200). WT mice were fed with control liquid diet (pair‐fed) or diet containing 5% alcohol for 4 weeks. The mRNA expression of GP96 and GRP78 was measured by RT‐PCR in livers (C) and isolated hepatocytes (E) and liver macrophages (n = 4‐10). (D) Protein level of GP96 and GRP78 in liver lysate was analyzed by western blotting (n = 6). (F) BMDMs were treated with 25 mM ethanol and mRNA expression of GP96 and GRP78 (n = 6). (G) The protein level of GP96 was analyzed at different time intervals (n = 3). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: EtOH, alcohol; comp, compensated; tpm, transcripts per million.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques: RNA Sequencing Assay, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Myeloid‐specific GP96 deficiency alleviates chronic alcohol‐induced liver injury. Female WT and M‐GP96KO mice were fed with isocaloric control liquid diet (pair‐fed) or 5% alcohol‐containing Leiber‐DeCarli diet (alcohol‐fed) for 4 weeks, and liver‐to–body weight ratio (A), serum levels of ALT (n = 10‐16) (B), and liver triglycerides (C) were analyzed. Representative photomicrographs of hepatic injury and steatosis assessed by H&E (D) and Oil Red O (E) staining in livers of mice of the indicated genotype (magnification × 100). Percentage of Oil Red O–positive area was quantitated by ImageJ (n = 3‐6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: EtOH, alcohol; wt, weight.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques: Staining

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Mice lacking myeloid‐specific GP96 exhibit altered lipid metabolism after 4 weeks of alcohol consumption. (A) PPAR‐α protein was detected in nuclear extracts of livers by western blot, and expression was quantitated and normalized to pair‐fed group (n = 4). TBP was used as loading control. The mRNA expression of genes involved in fatty acid β‐oxidation (CPT1a, ACOX1, LCAD, and MCAD) (B‐E) and lipogenesis (SREBPF1, SCD1, and FAS) (F‐H) was analyzed in WT and M‐GP96KO hepatocytes by RT‐PCR and compared with pair‐fed control (n = 3‐6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Myeloid‐specific GP96 deficiency prevents chronic alcohol‐induced endotoxin and pro‐inflammatory cytokine production. (A) Endotoxin level was measured in serum (n = 8‐12). (B) CyP2e1 was detected in liver microsomal fraction by western blot, and calnexin was used as an internal loading control (n = 3). Total RNA from liver tissue was subjected to quantitative RT‐PCR for analysis of pro‐inflammatory cytokines TNF‐α, IL‐6, MCP‐1, and IL‐1β; NLRP3; anti‐inflammatory markers IL‐10, TGF‐β, and ATF3 (C); and macrophage markers (n = 6‐10) (F). (D) Total liver protein level for TNF‐α was measured in tissue extracts of pair‐fed and alcohol‐fed mice by ELISA (n = 6‐10). (E) ATF3 protein level was analyzed by western blot in whole‐liver extracts using tubulin as an internal control (n = 3). The mRNA expression profile of (G) pro‐inflammatory, and (H) anti‐inflammatory and restorative macrophage markers, Trem2 and ATF3, were analyzed in liver macrophages of pair‐fed and alcohol‐fed mice (n = 5). (I) The mRNA level of pro‐inflammatory cytokines was analyzed in BMDMs isolated from WT and M‐GP96KO mice and stimulated with 100 ng/mL LPS for 2 hours (n = 9). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques: Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Isolation

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Loss of myeloid‐specific GP96 prevents LPS‐induced liver injury and inflammation. Female WT and M‐GP96KO mice were injected intraperitoneally with LPS (0.5 mg/kg body weight or saline (n = 4‐8). Serum was collected after 18 hours and subjected to analysis of ALT (A) and AST (B) and compared with the control group. (C) Livers were collected 2 hours after LPS injection, and liver cytokine mRNA was analyzed by RT‐PCR. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques: Injection, Reverse Transcription Polymerase Chain Reaction

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Inhibition of GP96 using specific inhibitor PU‐WS13 and siRNA reduces LPS‐induced pro‐inflammatory cytokine production. BMDMs were isolated from C57BL/6J mice and stimulated with LPS (100 ng/mL) for 2 hours and treated with PU‐WS13 (0.5 μM) either alone for 2 hours or before LPS for 1 hour. DMSO‐treated cells served as control group (n = 8). RT‐PCR was carried out to evaluate the expression of TNF‐α (A), IL‐6 (B), IL‐1β (C), and MCP‐1 (D) and compared with the untreated group. (E) Culture supernatant was evaluated for secreted TNF‐α by ELISA. (F) Murine macrophage RAW 264.7 cell line was transiently transfected with GP96 siRNA (100 nM) or negative control siRNA for 48 hours and stimulated with LPS for the final 6 hours. Secreted TNF‐α level was measured in culture supernatant by ELISA. Data are presented as mean ± SEM. *** P < 0.001, **** P < 0.0001. Abbreviation: ND, not detected.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques: Inhibition, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Schematic representation depicting pathophysiological significance of macrophage‐specific GP96 during chronic alcohol‐mediated liver inflammation and injury.

Article Snippet: Antibodies used were GP96 (SMC‐105; StressMarq Biosciences, Cadboro, Canada), GRP78 (CST‐3183; Cell Signaling Technology), peroxisome proliferator‐activated receptor alpha (PPAR‐α) (sc‐9000; Santa Cruz Biotechnology, Dallas, TX), CyP2e1 (AB1252; MilliporeSigma, Burlington, MA), calnexin (ab2295; Abcam, Cambridge, United Kingdom), C/EBP‐homologous protein (CHOP) (sc‐7351; Santa Cruz Biotechnology), ATF6α (sc‐166659; Santa Cruz Biotechnology), ATF4 (10835‐1‐AP; Proteintech, Rosemont, IL), ATF3 (sc‐81189; Santa Cruz Biotechnology), β‐actin (ab6276; Abcam), tubulin (ab6046; Abcam), and TATA‐box protein (TBP) (ab818, Abcam).

Techniques:

Journal: Molecular Metabolism

Article Title: TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion

doi: 10.1016/j.molmet.2018.01.016

Figure Lengend Snippet: TALK-1 channels are expressed in mouse and human δ-cells. (A) Mouse pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 mice). (B) Mouse pancreas section stained for TALK-1 (green), ER (GRP94, red), and SST (cyan). (C) Human pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 pancreata). (D) Human pancreas section stained for TALK-1 (green), ER (GRP94, red), and somatostatin (E) K2P currents recorded from WT and TALK-1 KO δ-cells ( N = 3 mice per genotype). (F) K2P currents recorded from human δ-cells expressing TALK-1 DN or control mCherry. ( N = 3 islet preparations); * P < 0.05, ** P < 0.005.

Article Snippet: Sections were stained using primary antibodies against somatostatin (Santa Cruz Biotechnology sc-7819: 1:250), TALK-1 (Novus Biologicals #NBP1-83071; 1:175) or TALK-1a (Antibody Verify AAS72353C; 1:250), glucagon (Proteintech #15954-I-AP: 1:500), and the endoplasmic reticulum marker GRP94 (Novus Biologicals #NB300-619; 1:100); secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit (Jackson Immunoresearch #711-546-152; 1:300), DyLight 650-conjugated donkey anti-goat (Thermo Fisher #SA5-10089; 1:250), and Cy3-conjugated donkey anti-mouse (Jackson Immunoresearch #715-166-150; 1:500).

Techniques: Staining, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion

doi: 10.1172/JCI126022

Figure Lengend Snippet: (A) IP followed by WB analysis of JAK1, STT3A, and ngPD-L1 tyrosine phosphorylation (4G10) in FLAG–ngPD-L1–SK-HEP-1 cells with or without exposure to IL-6 (20 ng/mL) and ruxolitinib (10 μmol/L) for 30 minutes. (B) JAK1 interacts with ngPD-L1 in ER lumen. Representative images of individual immunofluorescence staining of JAK1 and PD-L1 interaction in ER region in Hep 3B cells by Duolink assay. The red dots (JAK1/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker. (C) Schematic showing JAK1/PD-L1 interaction in the ER. IC, intracellular domain; TM, transmembrane domain; EC, extracellular domain. (D) Trypsin digestion of ER fractions with (group 3) or without (group 2) permeabilization in Hep 3B cells.

Article Snippet: After trypsinization, samples were analyzed by Western blotting using primary antibodies against IRE1α (3294; 1:2000; Cell Signaling Technology), HSP90B1 (catalog NBP2-42379; 1:3000; Novus Biologicals), or JAK1 (catalog 610231; 1:2000; BD Biosciences).

Techniques: Phospho-proteomics, Immunofluorescence, Staining, Fluorescence, Marker

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: 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, 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