Bay11 7082

Recombinant soluble human TGF-β1 and TWEAK were from Peprotech (Rocky Hill, NJ, USA). Recombinant soluble human TNF-α was obtained from eBioscience (San Diego, CA, USA). Purified anti-α-tublin and anti-human Vimentin (V9) monoclonal antibodies (mAbs) SB431542 and AG1478 were from Sigma Chemicals (St. Louis, MO, USA). Anti-human E-cadherin (HECD-1) was from Takara (Tokyo, Japan). N-cadherin and anti-EGFR mAbs were from BD Biosciences (San Jose, CA, USA). Anti-phospho-EGFR (pY845) mAbs was from abcam (Cambridge, UK). Anti-Smad2/3, anti-phospho-Smad2 (Ser465/467), anti-extracellular signal-regulated kinase (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-Akt, anti-phospho-NF-κB p65 (Ser536) polyclonal antibodies, and anti-ZO-1, anti- Jun N-terminal kinase (JNK), anti-phospho-JNK (Thr183/Tyr185), anti-phospho-Akt (Ser473), and anti-NF-κB mAbs were obtained from Cell Signaling Technology (Beverly, MA, USA). SB202190, SP600125, LY294002, and BAY11-7082 were from Wako Chemicals (Osaka, Japan). AZD6244 was from Selleckchem (Houston, TX, USA). Bronchial epithelial growth medium (BEGM) was purchased from Cambrex (East Rutherford, NJ, USA).

5-FU (Kyowa Hakko, Tokyo, Japan) was administered to mice intraperitoneally (i.p.) at a dose of 150 mg/kg either as a single dose or once every 7 days two times. Recombinant Ang-1, Thpo and CXCL12 were purchased from R&D Systems (Minneapolis, MN) and diluted with phosphate-buffer saline (PBS). The pharmacological inhibitors PD98059 and LY294002 were purchased from Merck Millipore (Darmstadt, Germany) and diluted with dimethyl sulfoxide (DMSO; Wako, Osaka, Japan). The NF-κB inhibitor BAY-11-7082 (Wako) was reconstituted in DMSO (Wako) as a 10 mM stock solution and diluted with PBS. In animal experiments, BAY-11-7082 was administered i.p. at a dose of 10 mg/kg and mice were sacrificed after 12 h. The Sp1 inhibitor Mithramycin A (MIT) was purchased from Cayman Chemical (Ann Arbor, MI) and diluted with ethanol (Wako). Tolfenamic Acid (TA) was purchased from Wako and mixed with corn oil (Sigma, St. Louis, MO). MIT was administered i.p. once every 2 days, and TA was administered via oral gavage also once every 2 days. Verapamil was purchased from Eisai (Tokyo, Japan) and diluted with sterile water. LPS (Escherichia coli 055:B5) was purchased from FUJIFILM (Osaka, Japan) and diluted with PBS. LPS injections were administered intravenously (i.v.) at a dose of 250 µg/kg.

Mouse monocyte/macrophage cell line, RAW264.7 (TIB-71, ATCC), were cultured in RPMI1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS: HyClone) and antibiotics. Cells were stimulated with LPS (1 to 1000 ng/ml) for the indicated time. In some experiments, cells were pretreated with the Rac1 inhibitor EHT1864 (Tocris), the NF-κB inhibitor BAY11-7082 (Wako Pure Chemical Industries), or the NADPH oxidase inhibitor diphenyleneiodonium (DPI: Sigma) for 2 h, then subjected to LPS. EHT1864 associates with Rac1 tightly and causes the release of bound GTP, thus prevents effector interaction.[31 (link)] BAY11-7082 is an irreversible inhibitor of IκB kinase α and phosphorylation of cytokine-inducible IκBα, thus inactivates NF-κB. DPI, an inhibitor of flavoenzymes, blocks NADPH oxidase activity.

Fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 (clone BM8), FITC-conjugated CD19 (clone 6D5), phycoerythrin (PE)-conjugated anti-Ly-6G (clone 1A8), biotin-conjugated anti-NK1.1 (clone PK136), peridinin chlorophyll protein/Cy5.5 (PerCP/Cy5.5)-conjugated anti-CD45 (clone 30-F11), and phycoerythrin-Cy7 (PC7)-conjugated anti-CD3ε (clone 145-2C11) monoclonal antibodies were purchased from Biolegend (San Diego, CA). LPS from Escherichia coli 0111:B4, poly I:C, and 2-DG were purchased from Sigma-Aldrich (St. Louis, MO). Imiquimod (R837) was purchased from Invivogen (San Diego, CA). Oligomycin was purchased from Merck Millipore (Billerica, MA). AICAR was purchased from Cell Signaling Technology (Danvers, MA). BAY 11-7082 was purchased from Wako Pure Chemical Industries (Osaka, Japan).