Pd98059

Anti‐α‐tubulin antibody (#T5168) were purchased from Sigma‐Aldrich (St. Louis, MO). PolyI:C, biotin‐PolyI:C, polydA:dT, NF‐κB inhibitor BAY11‐7082, and MAPK inhibitors including SB203580, PD98059, and SP600125 were from InvivoGen (San Diego, CA). Recombinant mouse IFN‐α and IFN‐β were from PBL Assay Science (Piscataway, NJ). Recombinant human IFN‐β was from R&D system (Minneapolis, MN). Primary antibodies against GAPDH (#5174), β‐Actin (#3700), pSTAT1 (Tyr701, #9167), STAT1 (#14994), pTBK1 (Ser172, #5483), TBK1 (#3504), pIRF3 (Ser396, #29047), IRF3 (#4302), peIF2α (Ser51, #3398), eIF2α (#5324), Flag (#14793), and HRP‐linked secondary antibodies anti‐rabbit IgG (#7074) and anti‐mouse IgG (#7076) were from Cell Signaling Technology (Danvers, MA). Anti‐PKR (18244‐1‐AP) was purchased from Proteintech (Wuhan, China), anti‐IFIT2 (sc‐398610) was from Santa Cruz (Santa Cruz, CA). The fluorescence secondary antibodies were purchased from LI‐COR (Lincoln, NE).

The ERK1/2 pathway specific inhibitor PD98059 (Peprotech, Rocky Hill, NJ) and the NF-kB pathway specific inhibitor Celastrol (Invivogen, San Diego, CA) were stored at -20° C. Prior to EHEC infection, the NCM460 cells were incubated with either PD98059 (50 μM) or Celastrol (100 nM) for 1h. The inhibitors were kept during the 5 h of infection.

Unless otherwise stated, all chemicals were purchased from Sigma (St Louis, MO, USA). Stock solutions of amitriptyline (100 mM), bupropion (100 mM), citalopram (10 mM), EGTA (50 mM), fluoxetine (10 mM), and N-acety-l-cysteine (NAC, 300 mM) were prepared in distilled water (DW). 5-fluorouracil (100 μg/mL), doxorubicin (2 mg/mL), paroxetine (20 mM), PD98059 (20 mM), SB203580 (20 mM), SP600125 (20 mM), taxol (1 mg/mL), tianeptine (100 mM), and Z-VAD-FMK (10 mg/mL, InvivoGen, San Diego, CA, USA) were dissolved in dimethyl sulfoxide (DMSO). The solutions were then diluted in culture medium to the working concentration. Where DMSO was used as a solvent, solution containing equivalent concentration of DMSO was used as a control. Final DMSO concentrations for chemicals indicated above were ~0.1% (v/v).
Dulbecco’s modified Eagle’s medium (DMEM/F12), DMEM, fetal bovine serum (FBS), and horse serum were purchased from Gibco-BRL (Gaithersburg, MD, USA). RPMI-1640 was purchased from Lonza (Walkersville, MD, USA). Total glutathione assay kit was purchased from Assay Designs (Ann Arbor, MI, USA). FLICA apoptosis detection kit was purchased from Immunochemistry Technologies (Bloomington, MN, USA). MitoSox was purchased from Invitrogen (Carlsbad, CA, USA). JC-1 mitochondrial membrane potential detection kit was purchased from Biotium Inc (Hayward, CA, USA).

To assess the effects of ERK, JNK, and p38 pathway on the airway disorders induced by LPS in our model, where indicated, mice were treated with the specific inhibitors of ERK (PD98059, invivogen, 10 mg/kg) or JNK (SP600125, invivogen, 20 mg/kg) or p38 (SB203580, invivogen, 5 mg/kg) respectively. The inhibitor was solubilized in 2% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). Treatments with inhibitors or DMSO alone were given intraperitoneally 1 h before and 2 h after the first LPS inoculation on day 35, and once a day successively from day 36 to day 41.

MDA-MB-231 or MCF-7 cells (2.5 × 10³ cells/well) were incubated in a chamber slide with complete medium for 24 h. The cells were treated with chemerin (80 nM) or SB525334 (TGF-β type I receptor inhibitor, 20 μM: Selleckchem, Houston, USA) in the presence of TGF-β (10 ng/mL) for 24 h. In addition, the cells were treated with chemerin (80 nM), ZSTK474 (PI3K inhibitor, 20 μM: Selleckchem), or PD98059 (MEK1/2 inhibitor, 20 μM: InvivoGen, San Diego, USA) in the presence of IGF-1 (40 ng/mL) for 24 h. The cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 buffer. After blocking with 2% goat serum in PBS, the cells were incubated with specific primary antibodies against β-catenin, E-cadherin, or SMAD2/3 overnight at 4 °C. After washing, the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. The slide was mounted using Vectashield mounting medium with DAPI (Vector Laboratories, CA, USA). The images were collected using a Zeiss LSM 700 confocal microscope.