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