Journal:

Article Title: Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-α and interleukin-6 (IL-6) secretion and CCL25 gene expression in mouse primary gingival cell lines: IL-6-driven activation of CCL2

doi:

Figure Lengend Snippet: (A-C) CCL2 and TNFα levels in cell-free supernatants 24 hr after stimulation with rIL-6, rIL-6 plus sIL-6R, rIL-6 plus sIL-6R plus sgp130, sgp130, or PBS; data from 3 independent sets of experiments showing an increase in CCL2 secretion from gingival cells stimulated with rIL-6 plus sIL-6R; rIL-6 plus sIL-6R plus sgp130; or sgp130 compared to PBS control cultures. (D) Neutralization of sgp130 by anti-gp130 antibody causes an increase in CCL2 secretion. (E) A gingival cell line stained with sgp130 indicated that about one-fifth of the cells express gp130. (F) Cell supernatants used for CCL2 assays had no detectable TNFα production.

Article Snippet: Reagents used in this study included ultrapure P. gingivalis LPS (Invitrogen; San Diego; CA) and E. coli LPS (Sigma); recombinant IL-6 (rIL-6) (<0.01 ng endotoxin per μg cytokine (eBioscience; San Diego, CA); sIL-6R (<0.0 EU endotoxin per 1 μg cytokine receptor) (R&D Systems; Minneapolis, MN); two preparations of sgp130: recombinant human sgp130 (<1.0 EU endotoxin per 1 μg receptor) (R&D Systems), and recombinant mouse sgp130/Fc chimera (<1.0 ng endotoxin per 1 μg receptor) (R&D systems).

Techniques: Control, Neutralization, Staining

Journal:

Article Title: Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-α and interleukin-6 (IL-6) secretion and CCL25 gene expression in mouse primary gingival cell lines: IL-6-driven activation of CCL2

doi:

Figure Lengend Snippet: Stimulatory effects on CCL2 secretion from gingival cell lines

Article Snippet: Reagents used in this study included ultrapure P. gingivalis LPS (Invitrogen; San Diego; CA) and E. coli LPS (Sigma); recombinant IL-6 (rIL-6) (<0.01 ng endotoxin per μg cytokine (eBioscience; San Diego, CA); sIL-6R (<0.0 EU endotoxin per 1 μg cytokine receptor) (R&D Systems; Minneapolis, MN); two preparations of sgp130: recombinant human sgp130 (<1.0 EU endotoxin per 1 μg receptor) (R&D Systems), and recombinant mouse sgp130/Fc chimera (<1.0 ng endotoxin per 1 μg receptor) (R&D systems).

Techniques:

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: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 2. The expression of selected receptors (CXCR4, Leptin R, EGFR, IL2R gamma, and gp130) on cell membrane of selected breast cancer cell lines. gp130 was the only receptor that is overexpressed in selected triple negative breast cancer (TNBC) cell lines, MDA 231wild type (WT), MDA 231 K, and MDA 468 cell lines. The expression of the receptors was detected using fluorescent-labeled monoclonal antibodies to the selected receptors and flow cytometry. Asterisks indicates significant difference to non-TNBC cell lines (one-way ANOVA, Tukey post-hoc test; p<0.05). Bar graph represents the mean values (n = 4), and the error bars show the standard deviation.

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Membrane, Labeling, Bioprocessing, Flow Cytometry, Standard Deviation

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 4. The effect of Individual and combinatorial silencing of selected proteins in JAK/STAT pathway on expression of selected antiapoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1). The mRNA expression was determined using RT-PCR in MDA 231 (A) and MDA 468 cells (B). In both cell lines, the drop in mRNA level of all three antiapoptotic proteins was significantly higher in cells exposed to JAK2/gp130 siRNA combination. Bar graphs represent the mean values (n = 3), and the error bars show the standard deviation. The effect of single and combinational silencing of gp130 and JAK2 on Bcl2 and Mcl-1 at protein level was confirmed by western blotting (C) in MDA 231 cells. β-actin served as endogenous protein. A similar trend was noted at the protein level (D). Expression of antiapoptotic related molecules was normalized to untreated cells (NT). Bar graph represents the mean values (n = 3), and the error bars show the standard deviation. Asterisks indicates significant difference compared to all other study groups (one-way ANOVA, Tukey post-hoc test; p < 0.05).

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

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

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 5. The effect of Individual and combinatorial silencing of selected proteins in JAK/STAT pathway on expression of selected pro-apoptotic proteins (PTEN and p53). The mRNA expression of the pro-apoptotic proteins was determined using RT-PCR in MDA-MB-231 (A) and MDA-MB-468 cells (B). The mRNA levels of both PTEN and P53 proteins showed the most significant increase in both cell lines after simultaneous silencing of JAK2/gp 130. Bar graphs represent the mean values (n = 3), and the error bars show the standard deviation. Th effect of single and silencing of gp130 and JAK2 on PTEN and P53 at protein level was confirmed by western blotting in MDA 231 cells (C). β-actin served as endogenous protein. A similar trend was noted at the protein level (D). Expression of pro-apoptosis proteins was normalized to untreated cells (NT). Bar graph represents the mean values (n = 3), and the error bars show the standard deviation. Asterisks indicates significant difference compared to all other study groups (one-way ANOVA, Tukey post-hoc test; p < 0.05).

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

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

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 9. The effect of silencing STAT3 and the individual and combinational silencing of JAK2 and gp 130 on STAT3 activation in MDA 231 cell line using differential Western Blotting on cytoplasmic and nuclear compartments. GAPDH and Histone H3 levels were analyzed as cytoplasmic and nuclear markers, respectively, which indicated effective isolation of the sub-cellular compartments. The bands representing STAT3 show total STAT3 levels (phosphorylated and non-phosphorylated). (A) Western blot showing the cytoplasmic expression level of STAT3 and p-STAT3. The expression of p-STAT3 was negligible in cytoplasm compared to the total STAT3 levels. (B) Bar graph represents the cytoplasmic expression level of STAT3 in different study groups (n = 3; error bars represent standard deviation). The STAT3 levels in cytoplasm dropped most significantly as a response to silencing STAT3. (C) Differential western blot shows the nuclear expression level of STAT3 and p-STAT3. The effect of silencing is apparent on both total STAT3 and the p-STAT3 levels. (D) Bar graph shows nuclear protein levels of p- STAT3 in different study groups (n = 3; error bars represent standard deviation). The drop in level of p-STAT in nucleus was most significant in cells exposed to JAK2/gp130 siRNA combination.

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Activation Assay, Western Blot, Isolation, Expressing, Standard Deviation

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: NS1 SC09 interacts with LRPPRC. (A) 293T cells were transfected with NS1 SC09 -Flag, NS1 JL89 -Flag, or empty vector. Cell lysates were pulled down using the antibodies against Flag tag. Proteins were then separated using SDS-PAGE and stained with silver, after which they were identified using LC/MS. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to HA-IP. (C) HeLa cells were co-transfected with LRPPRC-Flag and NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Twenty-four h later, cells were assessed for the localization of NS1, LRPPRC, and TOMM20. Cyan color indicated the colocalization of LRPPRC and TOMM20 and white color indicated the colocalization of LRPPRC, NS1, and TOMM20. Scale bar: 5 μm. It was representative of 20 cells. (D) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection, Plasmid Preparation, FLAG-tag, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: Summary annotated proteins identified to interact with NS1 SC09 -flag but not NS1JL89-flag and Vec-flag.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Virus, Binding Assay

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The key domain responsible for the interaction between NS1 SC09 and LRPPRC. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to HA-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: LRPPRC is a key factor that regulates the replication of IAVs through the autophagy pathway and is antagonized by NS1 SC09 . (A and B) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were infected with WSN at the MOI of 0.01. The supernatants were sampled at 0, 12, 24, and 48 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. Cell lysates at 0, 12, 24, and 48 h post-infection were analyzed using western blotting. (C and D) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were infected with WSN at the MOI of 5.0, in the presence or absence of rapamycin (20 μm) or 3-MA (5 mM). The supernatants were sampled at 24 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. The cell lysates were analyzed using western blotting. (E) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were transfected with mRFP-GFP-LC3B and then infected with WSN at the MOI of 0.1. Cells were analyzed to assess the formation of autophagosomes. Fluorescence signals indicated the expression of RFP and GFP (yellow color: incomplete autophagy, red color: complete autophagy). Scale bar: 10 μm. It was representative of 20 cells. (F) The graph shows the quantification of mRFP+GFP+ autophagosomes and mRFP+GFP- autolysosomes by taking the average number of dots in 20 cells (n=average number of dots in 20 cells). (G and H) 293T cells and LPRRPC KO cells were infected with WSN-NS1 SC09 or WSN at the MOI of 0.1. The supernatants were sampled at 24 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. Cell lysates were analyzed using western blotting. (I) 293T cells and LPRRPC KO cells were transfected with NS1 SC09 -HA or NS1 WSN -HA. 24 h post-transfection, cell lysates were analyzed using western blotting. Error bars indicate the SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Infection, Virus, Titration, Western Blot, Transfection, Fluorescence, Expressing

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The interaction between NS1 SC09 and LRPPRC results in initiation of BECN1-dependent autophagy. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (D) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-BECN1 antibody or control IgG. (E) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP. (F) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-PIK3C3 antibody or control IgG. (G) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: A proposed model for the pathway of autophagy induced by NS1 pdm09 . In the normal physiological state, LRPPRC interacts with the BECN1-BCL2 heterodimer, forming a stable complex in the mitochondria, thereby inhibiting BECN1-dependent autophagy. During A/(H1N1) pdm09 infection, the NS1 pdm09 promotes viral replication by enhancing autophagy which is mainly attributed to the inhibitory effect of NS1 pdm09 on the negative regulation of autophagy by LRPPRC. The interaction between NS1 pdm09 and LRPPRC competitively blocks the interaction of LRPPRC with BECN1-BCL2 heterodimer, resulting in degradation of BCL2 and increased recruitment of BECN1 by PIK3C3, and then induction of the initiation of autophagy.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Infection

Journal: The International journal of developmental biology

Article Title: Activated Notch1 is a stronger astrocytic stimulus than leukemia inhibitory factor for rat neural stem cells.

doi: 10.1387/ijdb.092869nr

Figure Lengend Snippet: Fig. 1. Cortical NSC respond to LIF and CNTF stimulation. Proliferating neural progenitors/NSC expressed Nestin (A,B) together with GP130 (A) or LIFRβ (B). In control cultures, no signal for phospho (p)STAT3 was detected (C). After addition of LIF, NSC show nuclear pSTAT3. Accordingly, in immunoblot assays, control (CTL) cells have non-phosphorylated STAT3, and 10 ng/ml CNTF or the indicated LIF concentrations induced phosphorylation of STAT3 (D). The transcriptional activation of Socs3 is only found in cells stimulated with LIF or CNTF (E). Images are representative of 3 independent experiments performed in duplicate. Scale bars represent 20 μm for A and 50 μm for B. -RT, RNA without reverse transcriptase.

Article Snippet: Other cell markers were immunodetected with the following antibodies: Nestin (Developmental Studies Hybridoma Bank), β-Tubulin-III (mouse or rabbit TUJ1, Covance), GFAP (rabbit, DAKO; rat, Zymed), pSTAT3 (Cell Signaling), Sox2 (Chemicon), GP130 (Santa Cruz Biotechnology) and LIFRβ (Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Phospho-proteomics, Activation Assay, Reverse Transcription

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 2. The expression of cell surface IL-6R and gp130 in MG-63 cultures af- ter different treatments. Semiconfluent osteoblast cultures were left untreated or treated with PMA or TNF- for 4 h, and the expression of cell surface IL-6R and gp130 were analyzed by flow cytometry. Closed his- tograms represent isotype antibody controls, and open histograms show either anti-IL-6R or anti-gp130 antibody-stained cells as indi- cated. The expression of IL-6R was reduced by both PMA and TNF-, whereas the level of gp130 was unaffected. The PMA-induced down-regulation of IL-6R expression was es- sentially abolished by staurosporine (stauro) and partially reversed by Galardin (Gal), whereas the suppressive effect of TNF- on IL-6R expression was substantially dimin- ished by UO126, a potent inhibitor of MEK1/MEK2.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Expressing, Flow Cytometry, Staining

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 5. The expression of cell sur- face IL-6R and gp130 in MG-63 cul- tures. Semiconfluent osteoblast cultures were left untreated or treated with differ- ent compounds for 4 or 24 h as indicated, and the expression levels of cell surface IL-6R and gp130 were analyzed by flow cytometry. Open histograms represent untreated cultures labeled with either an- ti-IL-6R or anti-gp130 antibody on each panel. Closed histograms show either iso- type antibody controls (panels labeled blank) or treated cultures (all other pan- els) labeled with either anti-IL-6R or anti- gp130 antibody as indicated in each panel. Note that the inhibitors of protein synthesis (cycloheximide (cyclohex)) and intracellular protein transport (brefeldin A (bref-A)) essentially diminished the ex- pression of both the IL-6R and gp130 on the cell surface after 4 h. Inhibition of transcription (actinomycin D (actino)) re- quired longer time periods to reduce the expression of either IL-6R or gp130.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Expressing, Flow Cytometry, Labeling, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 6. Western blot analysis of IL-6R and gp130 in MG-63 cells. Semiconfluent cell cultures were left untreated or treated with various compounds for different time periods as indicated, and the amounts of IL-6R (panels A and B, upper blots) and gp130 (panels A and B, lower blots) were analyzed. Cells were lysed, 700 g of protein of each cell lysate was incubated with either anti-IL-6R or anti-gp130 antibodies, and then immunocomplexes were precipitated and analyzed by Western blot. Membranes were immunoblotted with either anti-IL-6R or anti- gp130 antibodies. Transcription and translation inhibitors reduced the amounts of both IL-6R and gp130, whereas brefeldin A induced the accumulation of the low molecular mass forms of IL-6R (80 kDa) or gp130 (130 kDa) shown as var-IL-6R and var-gp130. Note that inhibi- tion of N-glycosylation by tunicamycin or treatment of samples with N-glycosidase F resulted in the 105-kDa band corresponding to gp130 (deglycos-gp130).

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Western Blot, Incubation, Glycoproteomics

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 7. Western blot analysis of tyrosine phosphorylation of gp130 in MG-63 cells (panel A) and bone marrow-derived pri- mary human osteoblasts (panel B). Semiconfluent cell cultures were left untreated or treated with various compounds as indicated. Cells were lysed, 700 g of protein was incubated with anti-gp130 antibody, and then immunocomplexes were precipitated and analyzed by Western blot. Membranes first were immunoblotted with anti-phosphotyrosine antibody (4G10, upper blot on each panel) and then stripped and re- blotted with anti-gp130 antibody (lower blot on each panel). Note that IL-6 and sIL-6R added together or conditioned medium (CM) of PMA- treated cultures induced tyrosine phosphorylation of gp130 (p-gp130). This effect was abolished by neutralizing antibodies to IL-6R. Addition- ally, note that two forms of gp130 can be detected (gp130 and var- gp130) and only the 145-kDa gp130 form was tyrosine-phosphorylated.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Western Blot, Phospho-proteomics, Derivative Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Shedding of the Interleukin-6 (IL-6) Receptor (gp80) Determines the Ability of IL-6 to Induce gp130 Phosphorylation in Human Osteoblasts

doi: 10.1074/jbc.m200546200

Figure Lengend Snippet: FIG. 8. Upper panel, analysis of IL-6, IL-6R, and gp130 mRNA expression by RPA is shown. Semiconfluent cultures of osteoblasts were left untreated or were treated with various compounds for different time periods as indicated. RNA was isolated from each sample, and RPA was performed. Constitutive mRNA expressions were observed for both IL-6R and gp130. mRNAs encoding IL-6, LIF, and GM-CSF were increased in TNF-- or LPS-treated cultures after 24 h. Lower panels, reverse transcription-PCR analysis of the IL-6R. Nucleotide sequence on panel C represents a segment (1381–1860 bp) of IL-6R (gp80, GenBank accession nos. X12830 and M20566) including the transmembrane encoding region (uppercase letters) and the 94-base pair deletion (exon 2, underlined part, missing in the 125-bp-long PCR product on panel B) of the alternative spliced variant (shown as DS-sIL-6R) (5). Boldface lowercase letters represent either the sense (1402–1421) or complementary antisense (1600–1620) sequences of primers used for PCR reactions. The boldface and underlined codons show the termination sites for the cognate (TAG) and the spliced variant (TGA) IL-6R mRNA.

Article Snippet: Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 g of cell lysate protein was incubated with either 3 g of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 g of antihuman-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C.

Techniques: Expressing, Isolation, Reverse Transcription, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Schematic illustration showing the generation of engineered decoy EVs at the cellular level. Producer cells are genetically modified to express cytokine receptors without the signalling domain fused to an EV sorting domain for efficient display of cytokine receptors on the surface of the secreted EVs (decoy EVs), which can decoy cytokines specifically. B) and C) Schematic illustrations of the evolution of cytokine receptors to facilitate EV surface display and assessment of various designs in a cytokine induced reporter cell system for high throughput screening of TNFR1 and IL6ST decoy EVs. D) and E) List of various TNFR1 or IL6ST sorting domain fusions assessed in the initial screen. F) Engineered decoy EVs displaying TNFR1 purified from HEK293T cells transfected with the constructs encoding the different display constructs (Figure1D) evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. Data were normalized to control cells treated with TNFα only (5 ng/ml). G) Engineered EVs displaying IL6ST purified from HEK293T cells transfected with constructs encoding the different display constructs evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). F, G , Error bars, s.d. ( n = 3), **** P < 0.0001, *** P < 0.001, ** P < 0.01, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Genetically Modified, High Throughput Screening Assay, Purification, Transfection, Construct, In Vitro, Activation Assay, Control

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Schematic illustration showing the evolution of TNFR1 design by addition of trimerization domains to enhance loading and binding efficiency of the EV- displayed decoy receptors. B) and C) Systematic comparison of various TNFR1 designs with multimerization domains. Engineered EVs displaying TNFR1 purified from HEK293T cells transfected with constructs encoding TNFR1 multimerization sorting domain fusion proteins (as listed in ) evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. Data were normalized to control cells treated with TNFα only (5 ng/ml). D) Schematic illustration showing the evolution of IL6ST designs by addition of different multimerization domain to enhance loading and binding efficiency of displayed decoy receptors on EVs. E) and F) Engineered EVs displaying IL6ST purified from HEK293T cells transfected with constructs encoding IL6ST multimerization sorting domain fusion constructs (as listed in ) respectively evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). B, C, E, F , Error bars, s.d. ( n = 3), **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Binding Assay, Comparison, Purification, Transfection, Construct, In Vitro, Activation Assay, Control

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Engineered EVs displaying IL6ST purified from MSC cells stably expressing the optimised IL6STΔ-LZ-NST display construct, evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). B) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing the optimized TNFR1ΔΔ-FDN-NST display construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml) treated cells. C) WB of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl cells and EVs indicating the presence of classical EV markers; ALIX (96 kDa), TSG101 (44 kDa) and absence of Calnexin (67 kDa) in the isolated EVs. The WB results further demonstrate the presence of respective His-tagged decoy receptors; TNFR1ΔΔ-FDN-NST (48 kDa) and IL6STΔ-LZ-NST (94 kDa) both on cells and EVs. D) Transmission electron microscopy of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl-EVs with nanogold labelled antibody staining of respective decoy receptor indicated by white arrows. E) Schematic illustration showing the workflow of the multiplex bead-based flow cytometry assay. Isolated EVs incubated with up to 39 different bead populations coated with different capture antibodies, which are distinguishable by flow cytometry due to their different fluorescence intensities. EVs captured by the different beads are detected with detection antibodies either against PAN (CD63-APC, CD81-APC and CD9-APC), mIL6ST-APC, or hTNFR1-APC. F-H) Characterization of EV surface protein composition by using F) anti-PAN (CD63, CD81 and CD9), G) anti-hTNFR1 and H) anti-mIL6ST detection antibodies in multiplex bead-based assays to confirm marker co-expression on MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound to respective capture beads and upon using APC labelled detection antibody. A, B , Error bars, s.d. ( n = 3), **** P < 0.0001, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Purification, Stable Transfection, Expressing, Construct, In Vitro, Activation Assay, Control, Isolation, Transmission Assay, Electron Microscopy, Staining, Multiplex Assay, Flow Cytometry, Incubation, Fluorescence, Marker

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: Comparison of A) inhibitory dose response curves and B) calculated IC50 values (95% Confidence interval) for TNFα sequestration by HEK293T TNFR1ΔΔ-FDN-NST EVs and Etanercept. HEK293T NF-kB reporter cells were challenged with 10 ng/ml (1 ng) of TNFα along with increasing doses of either HEK 293T TNFR1ΔΔ-FDN-NST EVs or Etanercept. C) Effect of HEK293T TNFR1ΔΔ-FDN-NST EVs and IL6STΔ-LZ-NST EVs on TNFα levels in conditioned medium determined by ELISA at 6 hours post LPS stimulation of RAW 246.7 macrophages. Data were normalized to control cells treated with LPS only. D) Survival curve of LPS (15 mg/kg) induced systemic inflammation in mice treated with intravenous injection of either 1×10 HEK 293T TNFR1ΔΔ-FDN-NST EVs ( n =3) or 2×10 HEK293T IL6STΔ-LZ-NST EVs ( n =4) or PBS ( n =5) 3 hours post induction. E) Survival curve of LPS (15 mg/kg) induced systemic inflammation in mice treated with intravenous injection of either 1×10 MSC TNFR1ΔΔ-FDN-NST EVs or 1×10 MSC Ctrl EVs or 160 µ g Etanercept 3 hours post LPS induction. F) Percent relative bodyweight to initial bodyweight over time of mice induced with LPS (15 mg/kg). Mice were treated with intravenous injection of either 3.25×10 MSC IL6STΔ-LZ-NST EVs + 3.25×10 MSC TNFR1ΔΔ-FDN-NST EVs ( n =6), 6.5×10 MSC TNFR1ΔΔ-FDN-NST EVs ( n =6), 6.5×10 MSC IL6STΔ-LZ-NST EVs ( n =6), 6.5×10 MSC Ctrl EVs ( n =6), or PBS ( n =6) 3 hours post induction. A , Error bars, S.E.M ( n =3), **** P < 0.0001, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose. F , Error bars, S.E.M ( n = 6), **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective observation time.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Control, Injection

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A ) Description of the treatment protocol for TNFα decoy EVs in EAE. B) Clinical score (EAE-score, see Supplementary table 1) of disease progression over time and C) EAE-score at endpoint (day 16) in mice induced with EAE using MOG 35-55 peptide and treated with subcutaneous (S.C) administration of either 4×10 MSC TNFR1ΔΔ-FDN-NST EVs ( n =5), MSC Ctrl EVs ( n =5), or saline ( n =5) (on day 7, 10 & 13). D) Schematic description of treatment protocol for IL6 decoy EVs in EAE. E) Clinical score of disease progression over time and F ) EAE-score at endpoint (day 16) in mice induced with EAE using MOG 35-55 peptide and treated with intravenous (I.V) administration of either 5×10 MSC IL6STΔ-LZ-NST EVs ( n =5), MSC Ctrl EVs ( n =5), or saline ( n =6) (on day 1,3,5,7,8,9 & 11). G) Schematic description of treatment protocol for IL23 decoy EVs in EAE. H) Clinical score of disease progression over time and I) EAE-score at endpoint (day 16) in mice induced with EAE using MOG 35-55 peptide and treated I.V with either 1×10 HEK293TIL23B-LZ-NST EVs pre symptomatic ( n =5) (on day 5, 7 & 10), 6×10 HEK293TIL23B-LZ-NST EVs post symptomatic ( n =5) (on day 13), or saline ( n =6). C, F, I , Error bars, SEM *** P < 0.001, ** P < 0.01, * P < 0.05 statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response to mock treated animal.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Biomarker Discovery, Saline

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Imaging flow cytometry analysis (IFCM) with dot plots and example event images in the double positive (DP) gate of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. PBS + antibodies were used for background adjustment and for determining the gating strategy. B) Percentage of detected events positive for either hTNFR1 or mIL6ST or both in Imaging flow cytometry analysis of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. Percentage values determined from objects/ml in different gates. C) Transmission electron microscopy of double decoy EVs with nanogold labelled antibody staining of hTNFR1 (10 nm) and mIL6ST (5 nm). D) Multiplex EV surface characterization of PAN (CD63, CD81, and CD9) positive, hTNFR1 positive and mIL6ST positive population in MSC double decoy EVs and MSC Ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound different capture beads and upon using APC labelled detection antibody. E) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing either the optimized TNFR1ΔΔ-FDN-NST display construct or TNFR1ΔΔ-FDN-NST and IL6STΔ-LZ-NST construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing either the IL6STΔ-LZ-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml). F) Engineered EVs displaying IL6ST purified from MSC cells stably expressing either the optimized IL6STΔ-LZ-NST display construct or TNFR1ΔΔ-FDN-NST, evaluated for IL6/sIL6R decoy in an in vitro cell assay responsive to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing either the TNFR1ΔΔ-FDN-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). G) Schematic of the treatment protocol for double decoy EVs in TNBS induced colitis. H) Percent change in relative bodyweight to initial weight over the disease course and I) survival curve in mice induced with colitis by intrarectal injection of TNBS and treated I.V with either 3×10 MSC double decoy EVs ( n =13), 3×10 MSC double decoy EVs ( n =15), 10 µ g Tocilizumab and 1 µ g Etanercept ( n =14), or saline (n=15) 24 hours post disease induction. E, F , Error bars, s.d. ( n =3). **** P < 0.0001 statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose. H , Error bars, SEM. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Saline treated animal.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Imaging, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy, Multiplex Assay, Fluorescence, Purification, Stable Transfection, Expressing, Construct, In Vitro, Activation Assay, Control, Injection, Saline

Journal: Journal of neuropathology and experimental neurology

Article Title: Effects of oncostatin M on human cerebral endothelial cells and expression in inflammatory brain lesions.

doi: 10.1093/jnen/60.11.1087

Figure Lengend Snippet: Fig. 2. HCEC transcribe mRNA for gp130, OSMR-b, LIFR, and IL-6R, but not for CNTFR. RT-PCR was performed on RNA extracted from HCEC and reverse-transcribed (RT1) using oligo(dT) primers. RNA samples not submitted to reverse transcription were used as negative controls (RT2). PCR results for gp130, LIFR, and OSMR-b are shown in the upper panel. Note that the expression level of LIFR was below that of gp130 and OSMR-b even when a 3-fold (33) higher amount of cDNA was used as starting material in the RT-PCR. PCR amplicons of CNTFR, IL-6R and b-actin (b-A), which was used as an internal control, are shown in the lower panel. RNA extracted from early postmortem human brain (hu Brain) served as a positive control for CNTFR. Identical results were obtained in all 5 HCEC preparations analyzed. Length of bands from DNA size marker (M, 1 kb DNA ladder, Gibco BRL) is indicated on the left (A). Surface detection of gp130 and OSMR-b, but not of LIFR, on HCEC. FACS analysis was performed as described in Materials and Methods to determine surface expression of gp130, OSMR-b, and LIFR on HCEC. Histograms show stain- ing with the respective receptor antibody (shaded) underlaid with the isotype-matched control (single line). Hela cells were

Article Snippet: Human recombinant IL-6, LIF, OSM, and anti-human gp130 and LIFR neutralizing antibodies were obtained from R & D Systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Expressing, Control, Positive Control, Marker, Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: Effects of oncostatin M on human cerebral endothelial cells and expression in inflammatory brain lesions.

doi: 10.1093/jnen/60.11.1087

Figure Lengend Snippet: Fig. 5. OSM-induced upregulation of IL-6 is mediated via gp130. HCEC were preincubated with increasing concentrations (0.5–50 mg/ml) of neutralizing antibodies against gp130 or an isotype-matched control antibody (mouse IgG1k, 50 mg/ml) for 2 h at 378C. Subsequently, OSM (100 ng/ml) was added to cells for 20 h. Protein concentrations of IL-6 in supernatants were analyzed by ELISA. Results are expressed as the percentage of IL-6 production compared to the isotype control. Data are the mean 6 SEM from 3 experiments. *p , 0.05, ***p , 0.001.

Article Snippet: Human recombinant IL-6, LIF, OSM, and anti-human gp130 and LIFR neutralizing antibodies were obtained from R & D Systems (Minneapolis, MN).

Techniques: Control, Enzyme-linked Immunosorbent Assay