Journal: International Journal of Molecular Sciences

Article Title: Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130

doi: 10.3390/ijms25031363

Figure Lengend Snippet: sgp130Fc inhibits IL6, IL11, OSM, and CT1-induced STAT3 activation. ( A ) ELISA of IL6R, IL11RA, LIFR, and OSMR in the conditioned supernatant of A549, primary human hepatocytes (HEP), and primary human hepatic stellate cells (HSC) 17 h after plating. Data are shown as box-and-whisker plots with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers). Western blots of p -STAT3 and STAT3 in CNTF, CT1, IL6, IL11, LIF (5 ng/mL)-stimulated ( B ) A549, ( C ) HEP, ( D ) HSC (15 min) and ( B – D ) immunoblots of p -STAT3, STAT3, GAPDH in OSM (5 ng/mL)-stimulated A549, HEP, HSC (15 min) in the presence or absence of sgp130Fc (5 µg/mL). ( A – D ) n = 4 biological replicates. BL: baseline. n.d. not detected.

Article Snippet: Recombinant human (rh)CNTF (257-NT, R&D Systems, Minneapolis, MN, USA), rhCT1 (612-CD, R&D Systems, Minneapolis, MN, USA), rhIL6 (206-IL, R&D Systems, Minneapolis, MN, USA), rhIL11 (Z03108, Genscript, Minneapolis, MN, USA), rhLIF (7734-LF, R&D Systems, Minneapolis, MN, USA), rhOSM (PHC5015, ThermoFisher Scientific, San Francisco, CA, USA), sgp130Fc (671-GP, R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Whisker Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130

doi: 10.3390/ijms25031363

Figure Lengend Snippet: Dose-dependent inhibition of IL6 and OSM cis -signalling by sgp130Fc. ( A ) Western blots of STAT3 activation status ( p -STAT3/STAT3) in A549 and HEP in the following conditions: without stimulus (BL), IL6 + PBS, or IL6 + sgp130Fc in the presence of either DMSO or GW280264X. ( B ) ELISA of IL6R in the supernatant of A549 after 17 h incubation with either DMSO or GW280264X. Data are shown as box-and-whisker plots with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers); 2-tailed Student’s t -test. ( C ) Western blots and ( D ) densitometry analyses of p -STAT3/STAT3 in GW280264X-treated A549 following stimulation with IL6 in the presence of different concentrations of sgp130Fc. ( E ) Western blots of p -STAT3, STAT3, and GAPDH, and ( F ) densitometry analyses of p -STAT3/STAT3 in OSM-stimulated A549 in the presence of increasing concentrations of sgp130Fc. ( G ) Western blots and ( H ) densitometry analyses of p -STAT3/STAT3 in IL6-stimulated HEP and HSC in the presence of increasing concentrations of sgp130Fc. ( D , F , H ) Data are shown as mean ± s.d. ( A – H ) n = 4 biological replicates; DMSO (0.1%), GW280264X (1 µM), IL6 (5 ng/mL), OSM (5 ng/mL), sgp130Fc (5 µg/mL, unless otherwise specified). BL: baseline. n.d. not detected.

Article Snippet: Recombinant human (rh)CNTF (257-NT, R&D Systems, Minneapolis, MN, USA), rhCT1 (612-CD, R&D Systems, Minneapolis, MN, USA), rhIL6 (206-IL, R&D Systems, Minneapolis, MN, USA), rhIL11 (Z03108, Genscript, Minneapolis, MN, USA), rhLIF (7734-LF, R&D Systems, Minneapolis, MN, USA), rhOSM (PHC5015, ThermoFisher Scientific, San Francisco, CA, USA), sgp130Fc (671-GP, R&D Systems, Minneapolis, MN, USA).

Techniques: Inhibition, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130

doi: 10.3390/ijms25031363

Figure Lengend Snippet: Examples of pre-clinical and clinical studies where sgp130Fc was given at doses that exceeded its half-maximal inhibitory (IC 50 ) concentrations for IL6 or OSM cis -signalling. Dose per mouse reports published values or is estimated based on an average adult mouse mass of 25 g. Estimated maximum concentrations for mouse studies were calculated based on the assumption that sgp130Fc is confined and equally distributed across the extracellular fluid spaces (interstitial fluid and plasma), which represents ~20% of the body weight of an adult mouse [ 20 ]. i.p.: intraperitoneal; i.v.: intravenous, d: day.

Article Snippet: Recombinant human (rh)CNTF (257-NT, R&D Systems, Minneapolis, MN, USA), rhCT1 (612-CD, R&D Systems, Minneapolis, MN, USA), rhIL6 (206-IL, R&D Systems, Minneapolis, MN, USA), rhIL11 (Z03108, Genscript, Minneapolis, MN, USA), rhLIF (7734-LF, R&D Systems, Minneapolis, MN, USA), rhOSM (PHC5015, ThermoFisher Scientific, San Francisco, CA, USA), sgp130Fc (671-GP, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130

doi: 10.3390/ijms25031363

Figure Lengend Snippet: Comparison of the inhibitory effects of sgp130Fc versus a gp130 neutralising antibody on IL6 or OSM cis -signalling.( A ) Western blots of p -STAT3, STAT3 (for IL6 and OSM), and GAPDH (for OSM) and ( B ) densitometry analyses of p -STAT3/STAT3 in GW280264X-treated A549 following stimulation with IL6, or OSM in the presence of either IgG, sgp130Fc, or a neutralising antibody against gp130 (clone B-R3). ( C ) Levels of IL6R, and OSMR protein in the supernatant from GW280264X-treated A549 as quantified by ELISA. ( D ) Schematic showing our proposed interpretation of the comparative effects between low versus high concentrations of sgp130Fc on the inhibition of gp130-mediated cis - and/or trans- signalling. ( A – C ) n = 4 biological replicates; B-R3 (5 µg/mL), GW280264X (1 µM), IgG (5 µg/mL), IL6 (5 ng/mL), IL11 (5 ng/mL), OSM (5 ng/mL), sgp130Fc (100 µg/mL). ( B , C ) Data are shown as box-and-whisker plots with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers). ( B ) One-way ANOVA with Tukey’s correction. BL: baseline; FC: fold change. n.d. not detected.

Article Snippet: Recombinant human (rh)CNTF (257-NT, R&D Systems, Minneapolis, MN, USA), rhCT1 (612-CD, R&D Systems, Minneapolis, MN, USA), rhIL6 (206-IL, R&D Systems, Minneapolis, MN, USA), rhIL11 (Z03108, Genscript, Minneapolis, MN, USA), rhLIF (7734-LF, R&D Systems, Minneapolis, MN, USA), rhOSM (PHC5015, ThermoFisher Scientific, San Francisco, CA, USA), sgp130Fc (671-GP, R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Whisker Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Regulation of adenosine system at the onset of peritonitis.

doi: 10.1093/ndt/gfp542

Figure Lengend Snippet: Fig. 5. sIL-6R is essential for A1R up-regulation both in vitro and in vivo. (A) PMCs were incubated with LPS (10μg/ml), sIL-6R (10ng/ml) or a combination of both. Six hours after incubation, cells were harvested and mRNA levels of A1R were analysed by real-time PCR. (B) Peritoneal lavage was performed 0, 3 or 6h following inoculation with E. coli. IL-6 and sIL-6R levels were determined by ELISA. (C–D) Mice were inoculated with E. coli and administered with (C) anti-mouse IL-6R (5μg/mice), (D) soluble gp130-Fc chimera (250ng/mouse) or isotope-matched control antibody. At 6h (upper panel) or 12h (lower panel) following inoculation, PMC were scraped from the peritoneal surface and analysed for A1R mRNA levels or A1R protein levels, respectively. Results are presented as densitometry of protein blot depicted above. A1R mRNA and protein levels were normalized to β-actin. Results are expressed as mean ± SEM of three experiments, fold from CT, n = 5 for each experiment. *P < 0.05, **P < 0.01 from CT.

Article Snippet: Recombinant human IL-6, recombinant human sIL-6R, anti-mouse IL6R and soluble gp130-Fc chimera (sgp130-Fc) were purchased from R&D Systems (Minneapolis, MN).

Techniques: In Vitro, In Vivo, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Regulation of adenosine system at the onset of peritonitis.

doi: 10.1093/ndt/gfp542

Figure Lengend Snippet: Fig. 6. Suggested regulation of adenosine system during onset of peritonitis. (A) Bacterial stimulus up-regulates both CD39 and CD73 and down- regulates ADA and AK which results in high levels of adenosine. (B) sIL-6R shed from recruited neutrophils binds to IL-6 and induces A1R mRNA and protein expression after binding to the signaling molecule gp130. (C) Activation of A1R by low levels of adenosine enhances production of local pro- inflammatory cytokines and promotes leukocyte migration.

Article Snippet: Recombinant human IL-6, recombinant human sIL-6R, anti-mouse IL6R and soluble gp130-Fc chimera (sgp130-Fc) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Binding Assay, Activation Assay, Migration