1 mM nonessential amino acids, and 1 mM sodium pyruvate (Life Technologies, Grand Island, NY)]. All cultures were maintained at 37 °C in a humidity-controlled incubator with 5% CO2 and were grown to confluence over 5–6 days before addition of pathogenic bacteria C. rodentium. The cells were washed and placed in antibiotic-free medium for 1 h. Confluent stock monolayers were subcultured by trypsinization. In this study, we utilized mouse intestinal epithelial cell line CMT93 to better elucidate cell

signaling responses to enteric pathogens in vitro. Nine experiments were conducted independently with similar results. To determine the time-dependent intracellular changes of NF-κB and Smad 7 in response to pathogen exposure, CMT93 cells were exposed with Cr (2.5 × 107 CFU per well) for 1 h BMN 673 cost in Dabrafenib supplier antibiotic-free DMEM at 37 °C. Subsequently, the media and cell lysates were collected at 0, 15, 30, 60, 90, and 120 min and 14 and 24 h postpathogen exposure. Cells were washed and lysed [(1% Triton-X-100 supplemented with 0.1 μM phenylmethylsulphonyl fluoride, 0.1 μM sodium orthovanadate, and Halt protease inhibitor (10 μL mL−1,

Pierce cat# 78410, Thermo Scientific, Rockford, IL)]. The lysates were kept at −80 °C for future Western blot analysis. The culture supernatants were stored at −20 °C for future measurement of TNF-α cytokine production. Total RNA was isolated from frozen colonic tissue (distal part of the colon) and treated CMT93 cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s protocol. First-strand cDNA was synthesized using 2 μg of extracted total RNA (Ready-to-Go kit; Amersham Pharmacia Biotech, Piscataway, NJ). IL-10 and TGF-β colonic expression was determined by real-time PCR using QuantiTect SYBR green real-time PCR kit (Qiagen, Valencia, CA) on the Opticon II DNA thermocylcer (MJ Research, Waltham, MA). A PCR master mix was prepared according to the manufacturer’s PAK6 protocol with a reaction volume of 50 μL, using the following real-time cycler conditions: 95 °C for 15 min, 94 °C

for 15 s, 55 °C for 30 s, 72 °C for 30 s for 38 cycles. GAPDH was used as internal controls. LightCycler relative quantification software was used to normalize data to the same GAPDH mRNA level. Samples were run in duplicate. Mouse IL-10 and TGF-β commercially available PCR primers were purchased from Biosource International, Inc. (Camarillo, CA) for detection, while GAPDH commercially available upstream and downstream PCR primers were utilized for detection (R&D Systems, Minneapolis, MN). Mouse colonic tissue and treated CMT93 cells were homogenized with lysis buffer prepared as previously mentioned. The suspensions were centrifuged at 4 °C, and the supernatant was collected, and protein content was determined using DC protein assay (Bio-Rad Laboratories, Hercules, CA).