Journal: Oncotarget

Article Title: Hypoxia suppresses cylindromatosis (CYLD) expression to promote inflammation in glioblastoma: possible link to acquired resistance to anti-VEGF therapy

doi:

Figure Lengend Snippet: (A) Immunohistochemical analysis with anti-CYLD and anti-CA IX antibodies in GBM tissues. In representative photomicrographs from 3 GBM cases, arrows indicate areas with high CA IX and low CYLD expression. Scale bars indicate 500 μm. (B) U87MG cells were incubated under hypoxic conditions, after which mRNA expression of hypoxic markers (left panel) and CYLD mRNA (right panel) were determined via qPCR. CYLD protein expression was determined by using Western blotting 24 h after hypoxic culture (right panel). * P< 0.05. (C) Expression of IL-6 and IL-8 mRNA was determined via qPCR after CYLD knockdown by using siRNA. * P< 0.01. (D) mRNA expression of inflammatory cytokines (left panel) was determined with qPCR under normoxic and hypoxic conditions. Cells were incubated with 10 ng/mL TNF-α for the indicated periods under normoxic and hypoxic conditions, and then mRNA expression of IL-6, IL-1β, and TNF-α was determined by using qPCR (right panels). * P< 0.05 compared with normoxic conditions. Values are means ± SE of triplicate samples.

Article Snippet: Goat polyclonal anti-human IL-6 antibody and anti-mouse CD31/PECAM-1 antibodies were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Expressing, Incubation, Western Blot

Journal: Oncotarget

Article Title: Hypoxia suppresses cylindromatosis (CYLD) expression to promote inflammation in glioblastoma: possible link to acquired resistance to anti-VEGF therapy

doi:

Figure Lengend Snippet: (A) Western blotting to determine the relative amounts of FLAG-tagged exogenous (exo) CYLD protein versus endogenous (endo) protein. (B) Each stable cell line was incubated for the indicated periods under hypoxic conditions, after which mRNA expression of IL-6 (left panel) and IL-8 (middle panel) was determined via qPCR. * P< 0.05 compared with normoxic conditions. Values are means ± SE of triplicate samples. IL-6 protein level (right panel) in total cell lysates (Lysate) and conditioned medium (CM) under normoxic (N) and hypoxic (H) conditions was determined via Western blotting. (C) mRNA expression of various inflammatory cytokines in U87MG-vector and U87MG-CYLD cells under normoxic or hypoxic conditions was determined via qPCR. The dashed line indicates the expression level of each cytokine in U87MG-vector cells under normoxic conditions. * P< 0.05, † P< 0.01, § P< 0.001, and ‡ P< 0.0001 compared with U87MG-vector cells in normoxia unless otherwise indicated. (D) Cells were incubated with 10 ng/mL TNF-α for 2 h under normoxic or hypoxic conditions, and mRNA expression of IL-6 (left panel) and IL-1β (right panel) was determined via qPCR. The Y-axis shows the fold change in mRNA expression versus no treatment. * P< 0.0005. Values are means ± SE of triplicate samples.

Article Snippet: Goat polyclonal anti-human IL-6 antibody and anti-mouse CD31/PECAM-1 antibodies were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Stable Transfection, Incubation, Expressing, Plasmid Preparation

Journal: Oncotarget

Article Title: Hypoxia suppresses cylindromatosis (CYLD) expression to promote inflammation in glioblastoma: possible link to acquired resistance to anti-VEGF therapy

doi:

Figure Lengend Snippet: (A) mRNA expression of various inflammatory cytokines in U87MG-vector and U87MG-CYLD tumors treated with PBR or bevacizumab for 18 days was determined via qPCR. The dashed line indicates the expression level of each cytokine in U87MG-vector tumors treated with PBS. * P< 0.05, † P< 0.01, § P< 0.001, and ‡ P< 0.0001 compared with 87MG-vector tumors treated with PBS unless otherwise designated. (B) Heat maps representing gene expression changes observed in in vitro (left panel) and in vivo (right panel) experiments. (C) Kaplan-Meier plots of overall survival in each experimental group. * P< 0.05 compared with the PBS-treated U87MG-vector group; † P< 0.0001 compared with the PBS-treated U87MG-CYLD group; § P< 0.005 compared with the bevacizumab-treated U87MG-vector group (log-rank test).

Article Snippet: Goat polyclonal anti-human IL-6 antibody and anti-mouse CD31/PECAM-1 antibodies were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Plasmid Preparation, In Vitro, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Moderate Physical Activity as a Prevention Method for Knee Osteoarthritis and the Role of Synoviocytes as Biological Key

doi: 10.3390/ijms20030511

Figure Lengend Snippet: ( A – E ): IL-1β immunohistochemistry. ( A ) IL-1β immunolabeling was very weak in the synovium of Group 1; ( B ) IL-1β immunolabeling was very weak in the synovium of Group 2; ( C ) IL-1β immunolabeling was moderate in the synovium of Group 3; ( D ) IL-1β immunolabeling was weak in the synovial membrane of Group 4; ( E ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-1-immunolabeling identified among groups. For details, see the text. ( F – J ): IL-4 immunohistochemistry. ( F ) IL-4 immunolabeling was moderate in the synovium of Group 1; ( G ) IL-4 immunolabeling was strong in the synovium of Group 2; ( H ) IL-4 immunolabeling was weak in the synovium of Group 3; ( I ) IL-4 immunolabeling was strong in the synovial membrane of Group 4; ( J ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-4-immunolabeling identified among groups. For details, see the text. ( K – O ): IL-6 immunohistochemistry. ( K ) IL-6 immunolabeling was moderate in the synovium of Group 1; ( L ) IL-6 immunolabeling was strong in the synovium of Group 2; ( M ) IL-6 immunolabeling was very strong in the synovium of Group 3; ( N ) IL-6 immunolabeling was very strong in the synovial membrane of Group 4; ( O ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-6-immunolabeling identified among groups. For details, see the text. ( P – T ): IL-10 immunohistochemistry. ( P ) IL-10 immunolabeling was moderate in the synovium of Group 1; ( Q ) IL-10 immunolabeling was strong in the synovium of Group 2; ( R ) IL-10 immunolabeling was weak in the synovium of Group 3; ( S ) IL-10 immunolabeling was strong in the synovial membrane of Group 4; and, ( T ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-10-immunolabeling identified among groups. For details, see the text. In inserts are the image analyses by the software in which red color represents immunolabeling. ( A – D , F – I , K – N , P – S ): Objective lens, 20×; scale bars: 50 µm. Results were presented as the mean ± SD. ANOVA was used to evaluate the significance of the results. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: After blocking, the sections were incubated overnight at 4°C with rabbit polyclonal anti-IL-1β (ab9787; Abcam), diluted 1:100 in PBS (Bio-Optica); rabbit polyclonal anti-IL-4 (ab9622; Abcam), diluted 1:100 in PBS (Bio-Optica); goat polyclonal anti-IL-6 antibody (sc-1265; Santa Cruz Biotechnology Inc., Dallas, TX, USA) diluted 1:100 in PBS (Bio-Optica); rabbit polyclonal anti-IL-10 (ab34843; Abcam), diluted 1:100 in PBS (Bio-Optica); mouse monoclonal anti-TNF-α antibody (ab199013; Abcam ) diluted 1:100 in PBS (Bio-Optica); mouse monoclonal anti-MMP-13 antibody (sc-81547; Santa Cruz Biotechnology Inc.) diluted 1:100 in PBS (Bio-Optica); rabbit polyclonal anti-lubricin antibody (ab28484; Abcam), diluted 1:100 in PBS (Bio-Optica).

Techniques: Immunohistochemistry, Immunolabeling, Software

Journal: BMC Cancer

Article Title: IL-6 signaling by STAT3 participates in the change from hyperplasia to neoplasia in NRP-152 and NRP-154 rat prostatic epithelial cells

doi: 10.1186/1471-2407-1-19

Figure Lengend Snippet: Constitutive IL-6 expression by NRP-152 & NRP-154 cells. Confluent cells were harvested, fixed, and permeabilized as described in Materials & Methods. They were stained with goat anti-rat IL-6, then with fluorescent anti-goat Ig. Cells were analyzed on a FACScan for fluorescence in FL2. A, NRP-152 cells. B, NRP-154 cells. In both histograms, green = fluorescent anti-goat Ig only; purple = goat anti-rat IL-6 plus fluorescent anti-goat Ig. KS analysis revealed that the fluorescence intensity of the NRP-154 cells in the second peak in panel B were 3 to 5 times the fluorescence intensity of the control-stained cells (green peak in panel B; p < 0.01). A histogram from 1 experiment is shown here.

Article Snippet: Cells were blocked by incubation on ice in goat Ig (Sigma), 2 mg/ml, for 45 min. After washing twice, cells were incubated with 1 or 2 μg/10 6 cells in 100 μl biotinylated goat anti-human IL-6 receptor (ligand-affinity purified; R&D Systems) on ice for 45 min. After washing three times, cells were incubated with phycoerythrin-labeled streptavidin for 45 min on ice.

Techniques: Expressing, Staining, Fluorescence

Journal: BMC Cancer

Article Title: IL-6 signaling by STAT3 participates in the change from hyperplasia to neoplasia in NRP-152 and NRP-154 rat prostatic epithelial cells

doi: 10.1186/1471-2407-1-19

Figure Lengend Snippet: IL-6 expression of clones A4 (red) and D8 (green) of NRP-154 cells. Clones of NRP-154 cells were derived by limit dilution at 1 cell/well; calculated cloning efficiency was 17.8%. Clones were fixed, permeabilized, and stained with biotinylated anti-IL-6 (R&D Systems) plus streptavidin-phycoerythrin (Pharmingen), as described in Materials & Methods. Fluorescence in FL2 was analyzed on a FACScan. The IL-6 expression of the 16 clones was either high, like A4 (shown in red), or low, like D8 (shown in green). A4 cells are 8 times as fluorescent as D8 cells. The IL-6 expression of 2 of the 16 clones is shown for the sake of clarity. The fluorescence intensity of clone A4 was 10× that of clone D8 in this experiment (p < 0.001 by KS statistics). A histogram from 1 determination is shown here.

Article Snippet: Cells were blocked by incubation on ice in goat Ig (Sigma), 2 mg/ml, for 45 min. After washing twice, cells were incubated with 1 or 2 μg/10 6 cells in 100 μl biotinylated goat anti-human IL-6 receptor (ligand-affinity purified; R&D Systems) on ice for 45 min. After washing three times, cells were incubated with phycoerythrin-labeled streptavidin for 45 min on ice.

Techniques: Expressing, Clone Assay, Derivative Assay, Staining, Fluorescence

Journal: BMC Cancer

Article Title: IL-6 signaling by STAT3 participates in the change from hyperplasia to neoplasia in NRP-152 and NRP-154 rat prostatic epithelial cells

doi: 10.1186/1471-2407-1-19

Figure Lengend Snippet: IL-6 receptor is expressed on untreated NRP-152 & NRP-154 cells. Confluent cells were removed with citrate-saline buffer and stained with 2 μg/10 6 cells goat anti-IL-6 receptor (R&D Systems). Fluorescent anti-goat Ig was then added. Fluorescence in FL2 was analyzed using a FACScan. A , NRP-152 cells. The blue line is the fluorescence control; the red line is the fluorescence due to the IL-6 receptor on the cells. The anti-IL-6 receptor-stained cells were 30 times as fluorescent as the control stained cells (p < 0.001 by KS statistics). B , NRP-154 cells. The green line is the fluorescence control; the purple curve is the IL-6 receptor on the cells. The anti-IL-6 receptor-stained cells (second red peak) were 10 times brighter than the control-stained cells (green peak; p < 0.001 by KS statistics). The first red peak was comprised of cells that did not stain positive for the IL-6 receptor.

Article Snippet: Cells were blocked by incubation on ice in goat Ig (Sigma), 2 mg/ml, for 45 min. After washing twice, cells were incubated with 1 or 2 μg/10 6 cells in 100 μl biotinylated goat anti-human IL-6 receptor (ligand-affinity purified; R&D Systems) on ice for 45 min. After washing three times, cells were incubated with phycoerythrin-labeled streptavidin for 45 min on ice.

Techniques: Saline, Staining, Fluorescence

Journal: BMC Cancer

Article Title: IL-6 signaling by STAT3 participates in the change from hyperplasia to neoplasia in NRP-152 and NRP-154 rat prostatic epithelial cells

doi: 10.1186/1471-2407-1-19

Figure Lengend Snippet: Dexamethasone treatment inhibited IL-6 expression in NRP-152 but not NRP-154 cells. Cells were grown in charcoal-adsorbed serum-containing medium prior to beginning dexamethasone treatment (10 -6 M) for 2 days. Cells were harvested, fixed, and permeabilized, stained for IL-6, then analyzed for fluorescence in FL2, as described above. Panel A , NRP-152 cells. Panel B , NRP-154 cells. Panel C , NRP-154 clone A3 (high IL-6 – expressing. Panel D , NRP-154 clone G7 (low IL-6-expressing). All panels , purple line = no dexamethasone; red line in panels A & B = + dexamethasone; blue line in panels C & D = + dexamethasone.

Article Snippet: Cells were blocked by incubation on ice in goat Ig (Sigma), 2 mg/ml, for 45 min. After washing twice, cells were incubated with 1 or 2 μg/10 6 cells in 100 μl biotinylated goat anti-human IL-6 receptor (ligand-affinity purified; R&D Systems) on ice for 45 min. After washing three times, cells were incubated with phycoerythrin-labeled streptavidin for 45 min on ice.

Techniques: Expressing, Staining, Fluorescence

Journal: Chembiochem : a European journal of chemical biology

Article Title: Gucosamine-6-sulfamate Analogs of Heparan Sulfate as Inhibitors of Endosulfatases

doi: 10.1002/cbic.201000401

Figure Lengend Snippet: (A) Biochemical evaluation of inhibitors against Sulf-1 and Sulf-2 using 4-MUS assay. Bars: percent inhibition in the presence of 5 mM inhibitor relative to uninhibited controls (*3a and 3b show full inhibition at 5mM, and bars are labelled with half maximal biochemical activity, or IC50, values). (B) Selectivity of sulfamate inhibitors. GlcN-6-sulfamates 3a and 3b are more selective for the Sulfs than PARS and are more potent than general inhibitors 1 and 2 (Bars: percent activity in the presence of 500µM inhibitor relative to uninhibited control). (C) Anti-inflammatory activity of Inhibitors. Production of IL-6 by IL-1β stimulation is a proinflammatory response in arthritis. Data show this response is inhibited by up to 50% by sulfamate-based sulfatase inhibitors (Bars: percent the IL-6 response is attenuated in the presence of 200 µM inhibitor relative to uninhibited control in IL-1β stimulated fibroblast-like synoviocytes).

Article Snippet: Cell-free culture supernatants or human recombinant IL-6 standard (25 µL) was added to the plates and incubated for 2 hours followed by the addition of 1 µg biotinylated goat antihuman IL-6 detection antibody (R & D Systems) for 1 hour.

Techniques: Inhibition, Activity Assay