Journal: PLoS ONE

Article Title: The Bacterial Preparation OK432 Induces IL-12p70 Secretion in Human Dendritic Cells in a TLR3 Dependent Manner

doi: 10.1371/journal.pone.0031217

Figure Lengend Snippet: A) Combinations of blocking antibody to TLR4, MyD88 inhibitory peptide and chloroquine were used to decipher contributions of different TLR in IL-12p70 production by DC after OK432 stimulation. The DC were pre-treated with the indicated compounds for 30 minutes at 37°C before maturation was induced by OK432. Immature DC were given only IL-4 and GM-CSF and Cyto were stimulated with the Jonuleit cytokine cocktail (IL-1β, IL-6, TNF-α and PGE 2 ). Only treatment with chloroquine reduced the level of IL-12p70 produced after OK432 stimulation. Mean+SEM is shown for 4 donors. B) To verify the effect of TLR4 blockade, a blocking antibody against TLR4 was used at 9 µg/ml. This reduced the level of LPS (20 ng/ml) induced IL-12p70 production by 50%. Mean of n = 2. C) MyD88 inhibition effectively decreases CL097 mediated IL-12p70 production via TLR7/8 signaling. D) Chloroquine has profound effects on the maturation status of OK432 stimulated DC. Percent MFI of CD1a, HLA-DR, CD86 and CD40 after pre-treating the DC with chloroquine before OK432 stimulation compared to OK432 alone (set to 100 percent) is shown. One representative experiment shown of three performed.

Article Snippet: The maturation of the cells was performed for 24 hours with the Jonuleit cytokine cocktail [IL-1β, 10 ng/ml; IL-6, 1000 U/ml; TNF-α 10 ng/ml (all from Immunotools, Friesoythe; Germany) and PGE 2 , 1 μg/ml (Sigma-Aldrich, St. Louis, MO)] or with OK432 (Picibanil, Chugai Pharmaceutical Co. Ltd, Tokyo, Japan).

Techniques: Blocking Assay, Produced, Inhibition

Journal: Molecular Biology of the Cell

Article Title: Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation

doi: 10.1091/mbc.E16-05-0273

Figure Lengend Snippet: The neddylation inhibitor MLN4924 inhibits NF-κB signaling in intestinal epithelial cells. (A) Western blot of nuclear/cytoplasmic fractionation of Caco-2 cells treated with TNF-α (10 ng/μl) and increasing concentrations of MLN4924 for 1 h leads to deneddylation of Cul-1 and decreased p65 NF-κB in the nucleus, with actin and TATA-binding protein (TBP) as loading controls ( n = 3). (B) Luciferase assay in Caco-2 cells transfected with an NF-κB luciferase reporter plasmid and treated with TNF-α and IL-1β (10 ng/μl each) and increasing concentrations of MLN4924 leads to increased inhibition of NF-κB signaling ( n = 3). (C) mRNA expression of NF-κB target genes 2 h after treatment with TNF-α and IL-1β (10 ng/ml each) is inhibited by pretreatment (30 min) with 3 μM MLN4924 treatment in the presence of ( n = 3), * p < 0.05.

Article Snippet: To inhibit caspase activation, a general caspase inhibitor peptide (Z-VAD-FMK; 30 μM) or a caspase-3 inhibitor peptide (Z-DEVD-FMK; 30 μM) was given as a 30-min pretreatment to T84 cells plated on permeable polyester inserts before treatment with cytomix plus MLN4924 (1 μM; BD PharMingen, San Diego, CA) or cytomix plus Bay 11-7085 (30 μM).

Techniques: Western Blot, Fractionation, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Inhibition, Expressing

Journal: Molecular Biology of the Cell

Article Title: Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation

doi: 10.1091/mbc.E16-05-0273

Figure Lengend Snippet: The combination of proinflammatory cytokines with the neddylation inhibitor MLN4924 leads to increased barrier disruption. (A) TER of T84 cells on Transwell inserts during a 24-h time course with control, cytomix (10 ng/μl each of TNF-α, IL-1β, and IFN-γ), 1 μM MLN4924, or cytomix plus MLN4924 ( n = 3). (B) FITC-flux assay of T84 cells on Transwell inserts after a 24-h time course with control, cytomix, 1 μM MLN4924, or cytomix plus MLN4924 ( n = 3). (C) Luciferase assay detecting activation of caspase-3/7 of T84 cells treated with control, cytomix, 1 μM MLN4924, or cytomix plus MLN4924 for 24 h, * p < 0.05.

Article Snippet: To inhibit caspase activation, a general caspase inhibitor peptide (Z-VAD-FMK; 30 μM) or a caspase-3 inhibitor peptide (Z-DEVD-FMK; 30 μM) was given as a 30-min pretreatment to T84 cells plated on permeable polyester inserts before treatment with cytomix plus MLN4924 (1 μM; BD PharMingen, San Diego, CA) or cytomix plus Bay 11-7085 (30 μM).

Techniques: Flux Assay, Luciferase, Activation Assay

Journal: Molecular Biology of the Cell

Article Title: Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation

doi: 10.1091/mbc.E16-05-0273

Figure Lengend Snippet: Proinflammatory cytokines with the neddylation inhibitor MLN4924 lead to increased apoptosis, and NF-κB inhibition leads to increased barrier disruption. (A) TER of T84 cells on Transwell inserts during a 24-h time course with cytomix plus MLN4924 in the presence of 30 μM general caspase inhibitor (Gen cas in) peptide or a negative control (Neg) peptide ( n = 3). (B) TER of T84 cells on Transwell inserts during a 16-h time course with cytomix plus MLN4924 in the presence of 30 μM caspase-3 inhibitor (Cas 3 in) peptide or a negative control (Neg) peptide ( n = 3). (C) Influence of necroptosis inhibitor necrostatin-1 on permeability of T84 cells to a combination of cytomix and MLN4924 ( n = 3). (D) TER of T84 cells on Transwell inserts during a 24-h time course with control, cytomix (10 ng/μl each of TNF-α, IL-1β, and IFN-γ), 1 μM MLN4924, 30 μM Bay 11-7085, or the combination of either cytomix plus MLN4924 or cytomix plus Bay 11-7085 ( n = 3). * p < 0.05.

Article Snippet: To inhibit caspase activation, a general caspase inhibitor peptide (Z-VAD-FMK; 30 μM) or a caspase-3 inhibitor peptide (Z-DEVD-FMK; 30 μM) was given as a 30-min pretreatment to T84 cells plated on permeable polyester inserts before treatment with cytomix plus MLN4924 (1 μM; BD PharMingen, San Diego, CA) or cytomix plus Bay 11-7085 (30 μM).

Techniques: Inhibition, Negative Control, Permeability

Journal: Molecular Biology of the Cell

Article Title: Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation

doi: 10.1091/mbc.E16-05-0273

Figure Lengend Snippet: Neddylation inhibition increases disease severity in a TNBS model of colitis. (A) Treatment of C57/BL6 mice treated with 2.5% TNBS and MLN4924 s.c. at 3 mg/kg per day shows decreased survival in mice receiving TNBS plus MLN4924 (29 total mice). (B) Decreased colon length in mice receiving TNBS plus MLN4924 compared with TNBS alone. (C) Histological colonic sections of mice treated with or without TNBS and with or without MLN4924. (D) TUNEL staining in mouse colon sections from TNBS plus MLN4924 experiment. (E) Cleaved caspase 3 IF staining in mouse colon sections from TNBS plus MLN4924 experiment. (F) Analysis of tissue injury index in mice receiving a combination of TNBS plus MLN4924 in comparison to TNBS alone. (G) Increased IL-1 and IL-6 protein expression as detected by MesoScale in mice receiving TNBS plus MLN4924 compared with TNBS alone.

Article Snippet: To inhibit caspase activation, a general caspase inhibitor peptide (Z-VAD-FMK; 30 μM) or a caspase-3 inhibitor peptide (Z-DEVD-FMK; 30 μM) was given as a 30-min pretreatment to T84 cells plated on permeable polyester inserts before treatment with cytomix plus MLN4924 (1 μM; BD PharMingen, San Diego, CA) or cytomix plus Bay 11-7085 (30 μM).

Techniques: Inhibition, TUNEL Assay, Staining, Expressing

Journal: Molecular Biology of the Cell

Article Title: Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation

doi: 10.1091/mbc.E16-05-0273

Figure Lengend Snippet: Mechanistic model of the effect of MLN4924 on the NF-κB pathway and downstream apoptotic responses. Under normal conditions, IκB is unphosphorylated and binds NF-κB subunits, sequestering them in the cytoplasm. An inflammatory stimulus leads to the phosphorylation of IκB, which is recognized by the Cul-1-Nedd8-transducin repeat–containing E3 ubiquitin protein ligase (TRCP) complex, targeting IκB for polyubiquitination and degradation by the proteosome. NF-κB can then translocate into the nucleus, leading to the transcription of target genes, including antiapoptotic cascades. Pharmacological inhibition of Cul-1 neddylation using MLN4924 stabilizes cellular IκB levels, keeping NF-κB out the nucleus and leading to decreased transcription of antiapoptotic target genes and increased apoptosis.

Article Snippet: To inhibit caspase activation, a general caspase inhibitor peptide (Z-VAD-FMK; 30 μM) or a caspase-3 inhibitor peptide (Z-DEVD-FMK; 30 μM) was given as a 30-min pretreatment to T84 cells plated on permeable polyester inserts before treatment with cytomix plus MLN4924 (1 μM; BD PharMingen, San Diego, CA) or cytomix plus Bay 11-7085 (30 μM).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: The Molecular Balance between Receptor Tyrosine Kinases Tie1 and Tie2 Is Dynamically Controlled by VEGF and TNFα and Regulates Angiopoietin Signalling

doi: 10.1371/journal.pone.0029319

Figure Lengend Snippet: HUVEC were incubated with VEGF A or TNFα B for times up to 24 h as indicated before cell lysis and detection of cellular full-length Tie1 by immunoblotting. Blots were stripped and re-probed for full-length Tie2 and tubulin, as indicated. Full-length Tie1 and Tie2 in endothelial cells treated with VEGF or TNFα at the different times points was determined by immunoblotting in at least three independent experiments and quantitated by densitometric scanning. Data are means and SEM and presented as arbitrary units normalized to the levels in untreated cells within each experiment. Asterisks indicate significant effect compared with untreated cells (p<0.05, Student's ‘t’test). C VEGF and TNFα stimulate formation of the intracellular Tie1 fragment. HUVEC were incubated with VEGF or TNFα for 30 min or 24 h as indicated before cell lysis and detection of cellular Tie1 endodomain by immunoblotting with an antibody recognizing the Tie1 C-terminus. Blots were stripped and re-probed for tubulin, as indicated. D VEGF and TNFα stimulate release of Tie1 and Tie2 ectodomain from endothelial cells. HUVEC were untreated or stimulated with VEGF or TNFα for 30 min or 24 h, as indicated, before collection of culture medium, removal of cellular material by centrifugation and immunoprecipitation of Tie1 and Tie2 extracellular domains. Immunoprecipitates were resolved by SDS/PAGE and released extracellular domains detected by immunoblotting. The relative mobility of mass markers is shown in kDa.

Article Snippet: As indicated in Results, cells were treated with the following concentrations of reagents: PMA, 10 ng/ml (Merck, Nottingham, UK); VEGF, 100 ng/ml (PeproTech, London, UK); TNFα, 100 ng/ml (PeproTech, London, UK); Ang1 200 ng/ml (R&D Systems, Abingdon, UK), unless otherwise indicated.

Techniques: Incubation, Lysis, Western Blot, Centrifugation, Immunoprecipitation, SDS Page

Journal: PLoS ONE

Article Title: The Molecular Balance between Receptor Tyrosine Kinases Tie1 and Tie2 Is Dynamically Controlled by VEGF and TNFα and Regulates Angiopoietin Signalling

doi: 10.1371/journal.pone.0029319

Figure Lengend Snippet: A Cells were incubated with PMA for the times indicated in hours before cell lysis and detection of cellular Tie1 endodomain by immunoblotting with an antibody recognizing Tie1 carboxy-terminus, as indicated. Blots were stripped and re-probed with an antibody to Tie2 intracellular domain, as indicated. The positions of molecular mass markers in kDa are shown on the left of the blots. B To confirm the 55 kDa Tie2 immunoreactive fragment is derived from Tie2 endothelial cells were transfected with control siRNA (Scr) or siRNA recognizing Tie2, as indicated. 24 h post-transfection cells were incubated with or without PMA for 24 h before lysis and detection of Tie2 endodomain by immunoblotting. C VEGF stimulates Tie2 endodomain accumulation at 24 h. Endothelial cells were incubated with VEGF for 0.5 or 24 h before cell lysis and detection of cellular Tie2 intracellular domain. Membranes were stripped and reprobed for Tie1 intracellular domain. D Effects of γ-secretase inhibition on Tie1 and Tie2 endodomains formed in response to PMA. HUVEC were incubated with or without the γ -secretase inhibitor in the absence and presence of PMA for 0.5 or 24 h as indicated. Tie1 endodomain was detected in whole cell lysates by immunoblotting with an antibody recognizing Tie1 carboxy-terminus and blots were stripped and re-probed with an antibody to Tie2 intracellular domain as indicated. Effects of γ-secretase inhibition on Tie1 and Tie2 endodomains formed in response to VEGF (E) and TNFα (F). HUVEC were incubated with or without the γ -secretase inhibitor in the absence and presence of VEGF or TNFα for 0.5 or 24 h as indicated. Tie1 endodomain was detected in whole cell lysates by immunoblotting with an antibody recognizing Tie1 carboxy-terminus and blots were stripped and re-probed with an antibody to Tie2 intracellular domain as indicated. G Tie1 endodomain interacts with full-length Tie2. HUVEC were incubated with VEGF or TNFα for 0.5 or 24 h as indicated. Cells were lysed and Tie2 immunoprecipitated. Tie1 endodomain co-immunoprecipitating with Tie2 was detected by immunoblotting with an antibody recognizing Tie1 carboxy-terminus and blots were stripped and re-probed with an antibody to Tie2. The relative mobility of mass markers is shown in kDa.

Article Snippet: As indicated in Results, cells were treated with the following concentrations of reagents: PMA, 10 ng/ml (Merck, Nottingham, UK); VEGF, 100 ng/ml (PeproTech, London, UK); TNFα, 100 ng/ml (PeproTech, London, UK); Ang1 200 ng/ml (R&D Systems, Abingdon, UK), unless otherwise indicated.

Techniques: Incubation, Lysis, Western Blot, Derivative Assay, Transfection, Control, Inhibition, Immunoprecipitation

Journal: PLoS ONE

Article Title: The Molecular Balance between Receptor Tyrosine Kinases Tie1 and Tie2 Is Dynamically Controlled by VEGF and TNFα and Regulates Angiopoietin Signalling

doi: 10.1371/journal.pone.0029319

Figure Lengend Snippet: HUVEC were treated with VEGF A, PMA B or TNFα C for 0, 0.5 or 24 h to induce Tie1 and Tie2 ectodomain cleavage followed by 200 ng/ml Ang1 for 30 min. Cells were lysed and phosphorylated Tie2 detected by immunoblotting with anti-phospho-Tie2 (pTie2), blots were stripped and re-probed for Tie1, Tie2 and tubulin. Phosphorylated Tie2 in cells treated with VEGF, PMA or TNFα for 0, 0.5 or 24 h and activated with Ang1 was detected by immunoblotting in at least three independent experiments and quantitated by densitometric scanning. Data are means and SEM of phospho-Tie2 normalized to that in Ang1-activated control cells within each experiment. Asterisks indicates significant increase in Ang1-induced Tie2 phosphorylation (p<0.05, Student's ‘t’test). D Loss of Tie1 enhances Ang1-activation of Tie2. HUVEC were transfected with contraol siRNA (C) or siRNA targeting Tie1 (Tie1) as indicated. 24 h post-transfection cells were activated with Ang1 for 30 min before lysis and detection of phosphorylated Tie2 (pTie2), blots were stripped and re-probed for Tie1 and Tie2. The relative mobility of mass markers is shown in kDa.

Article Snippet: As indicated in Results, cells were treated with the following concentrations of reagents: PMA, 10 ng/ml (Merck, Nottingham, UK); VEGF, 100 ng/ml (PeproTech, London, UK); TNFα, 100 ng/ml (PeproTech, London, UK); Ang1 200 ng/ml (R&D Systems, Abingdon, UK), unless otherwise indicated.

Techniques: Western Blot, Control, Phospho-proteomics, Activation Assay, Transfection, Lysis