Cell cultures without bacterial infection served as controls. The procedures were performed according to the instruction manuals and post-infection cells with non-stained trypan blue staining were directly counted. Enzyme-linked immuno-sorbent assay (ELISA) for cytokines To determine the optimal dose and incubation time of various bacteria, bacteria (H. Selonsertib ic50 pylori and L. acidophilus) were cultured with MKN45 cells (MOI 1-100) in an antibiotic-free RPMI 1,640 medium (5 ml) containing 10% FBS at 35°C in micro-aerophilic conditions for up to 8 hours. In the experimental study, L. acidophilus

were added to MKN45 cells and Tucidinostat in vitro incubated for 8 hours under the same conditions. After PBS washing and removal of the bacilli, an equal volume of H. pylori was added and the cells were incubated for another 4 hours. The final culture supernatant was centrifuged at 12,000 rpm for 5 min to remove bacteria and cell debris. Concentrations of TNF-α, IL-8 (R & D System, Minneapolis, MN), and TGF-β1 (eBioscience, San Diego, CA) were measured by ELISA according to the manufacturer’s instructions. The absorbance of each micro-plate was read on a spectro-photometer using 450 nm as the primary wave length and 570 nm as the

reference wave length. All tests were done in triplicate. Preparation of cytoplasmic and nuclear extracts The MKN45 and AGS cells were pre-treated with L. acidophilus for 8 hours followed by various doses of H. pylori for 1 hour; then cytoplasmic and nuclear extracts were isolated by a Nuclear Extract Kit (Active Motif, Japan). selleck chemicals Briefly, cells were washed with ice-cold saline containing phosphatase inhibitors and pelleted. The cell pellets were then re-suspended in a hypotonic buffer and incubated for 15 min on ice. They were lysed by the addition of detergent and vortexed vigorously for 10 s. After the nuclei were pelleted and re-suspended in complete lysis buffer, the tube was vigorously shaken at 4°C for 30 min on a shaking platform. The nuclear extracts were then centrifuged and the supernatants were aliquoted and stored at -80°C. RT-PCR for cytoplasmic

Smad7 Total RNA was isolated from MKN45 cells using a commercial kit (ImProm-ll™ Reverse Transcription System, Promega, USA) after H. pylori and L. acidophilus MycoClean Mycoplasma Removal Kit incubation. The RNA was quantified by determining absorbance at 260 nm. One μg RNA was converted to cDNA, which was stored at -72°C until use. The human Smad7 primer sequences were forward 5′-CATCACCTTAGCCGACTCTG-3′ and reverse 5′GTCTTCTCCTCCCAGTATGC-3′, generating a 224 bp fragment [30]. For Jak1 and Stat1, the primer sequences were forward 5′-GCAGCCAGCATGATGAGA-3′ and 5′-GTGGACGAGGTTTTGTAAGGA-3′ and reverse 5′-CTCGGAAGAAAGGCCTCTG-3′ and 5′-CAGACACAGAAATCAACTC-3′, generating fragments of 607 bp and 518 bp, respectively [31, 32]. The PCR condition was as follows; 95°C for 5 min, followed by 25 cycle of 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min, and finally 72°C for 7 min.