Mek inhibitor

To determine the induction of cytokines expression, FACS-sorted T cells and mLN cells or splenocytes were cultured in 96-well plates in IMDM medium, 10% FCS, 1% l-glutamine, 1% penicillin/streptomycin (GIBCO), 5 × 10⁻⁵ M 2-mercaptoethanol in the presence or absence of Monoensin (Invitrogen) (10 μM) and rmIL-33 (PeproTech) (10 ng/mL) or anti-CD3 (BD, 145-2C11) (2 μg/mL) for six hours. Alternatively, intracellular amounts of p-EGFR (Y1068 and Y992), p-ERK and IκBα were analyzed after 30 minutes of stimulation with IL-33 (10 ng/mL), anti-CD3 (2 μg/mL), AREG (R&D) (10 ng/mL) or HB-EGF (R&D) (10 ng/mL). To analyze EGFR or AREG induction, splenocytes or FACS-sorted T cells were re-stimulated overnight with H. polygyrus excretory-secretory products (HES; Johnston et al., 2015 ) (1 μg/mL), adult worm extract (HEX) (10 μg/mL), anti-CD3 (2 μg/mL), IL-7 (PeproTech) (20 ng/mL), IL-2 (BD) (1 ng/mL), TSLP (eBiosciences) (20 ng/mL) and IL-33 (10 ng/mL). Inhibitors were used at the following concentrations: MEK inhibitor (Promega)(10 μM), Gefitinib (LC laboratories) (1 μM) and Marimastat (AbCam) (25 μM). The inhibitory effect of Marimastat (25 μM) was checked by its ability to prevent the release of activated Amphiregulin by transfected HEK cells. Under these conditions cell viability was higher than 90% as compared to 97% at the start of the experiment.

Primary HCEs (pHCEs) were plated in T25 flasks, grown to 70–80% confluence and starved overnight by incubating cells in basic K-SFM (without EGF and bovine pituitary extract [BPE] supplements). After starving the cells, they were treated with 2 ng/ml TGF-β1 (R&D Systems; Minneapolis, MN), 5 ng/ml EGF (R&D Systems), 10 μM MEK inhibitor (U0126; Promega; Madison, WI), or 30 μM EGFR inhibitor (Tyrphostin AG1478: Selleck Chemical LLC/Thermo Fisher Scientific; Pittsburgh, PA) alone or in combination for specified times at 37 °C in a humidified 5% CO₂ incubator. Cells in basic K-SFM served as controls. Cells were harvested for western blot (WB) or reverse transcriptase-polymerase chain reaction (RT-PCR).