Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Established PAR1-expressing MKN45 stable cell line MKN45 cells were transfected using LipofectAMINE2000 (Invitrogen) and pcDNA3.1-PAR1 (MKN45/PAR1) or pcDNA3.1-empty-vector alone (for MKN45/mock as the control). Individual G418 resistant (0.75 mg/ml) clones were picked and analyzed for PAR1 expression by RT- PCR and immunoblotting of total cell extract. Western blot Total cell protein was extracted using RIPA buffer. Pro- teins in the lysate were resolved by SDS-PAGE using a 5-20% SuperSep gel (Wako, Osaka, Japan). The resolved proteins were transferred to nitrocellulose membrane. Protein bands were incubated with primary antibody overnight at 4°C. Signals were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). Cell growth analysis To examine the in vitro cell growth rate, MKN45/mock, MKN45/PAR1 and MKN28 cells were seeded into 24- well plates at 1.0×104cells/well. Various cultures were incubated for different periods of time while being exposed to one or more of the following: PAR1 agonists, a-thrombin and TFLLR-NH2, and PAR1 antagonist, SCH79797 and resultant growth rates were quantified. The level of pro-thrombin was 1-2 μM, and a concen- tration of active thrombin in the 10 nM range was almost certainly physiologically relevant [26]. The TRAP analogue, TFLLR-NH2, can selectively activate PAR1 at concentrations lower than 50 μM [27]. Thus, we selected an a-thrombin concentration of 15 nM and a TFLLR-NH2 concentration of 30 μM to determine if these enzymes would stimulate proliferation of MKN45/ PAR1 and MKN28 cells. Cell numbers were counted with a hemocytometer at 24, 48, 72 and 96 hrs after seeding of cells. Cell invasion assay In addition to establishing that the activation of PAR1 in a gastric carcinoma cell background can stimulate cell replication, we wished to evaluate the ability of PAR1 to stimulate cell invasion. Invasion of cells through matri- gel was determined using a Transwell system (CHEMI- CON) as described previously [28]. a-thrombin was added at 15 nM, TFLLR-NH2 was added at 30 μM and SCH79797 was added at 35, 70, or 150 nM to the cells (0.5×106cells/well) in the upper well containing serum- free medium. After the addition of fresh medium con- taining 10% FBS to the lower chamber, incubation was continued for 24 hr at 37°C. The cells on the underside of the membrane were stained and dissolved in 10% acetic acid for measurement of A560 nm. The A560 nm of the MKN45/mock, MKN28 and MKN45/PAR1 cells Page 3 of 14 cultured under noted conditions were determined and compared using the A560 nm of MKN45/mock and MKN28 cultured under a PAR1 agonist-free condition as a baseline. Measurement of NF- B Activation by Electrophoretic Mobility Shift Assay MKN45/mock and MKN45/PAR1 were treated for 0.5, 1, 2, 6, 12, and 24 hr with 15 nM a-thrombin. Nuclear fractions were extracted from the cultured cells using NE-PER (PIERCE, Rockford, IL, USA). Assays were per- formed using an oligonucleotide with the NF- B motif, 5′-AGTTGAGGGGACTTTCCCAGGC-3′, which was labeled with biotin for chemiluminescence detection. Nuclear extracts of MKN45/PAR1 and MKN45/mock were isolated and a Gel mobility shift assay was per- formed by incubating each of the nuclear extracts with the labeled probe and competing oligonucleotides in binding buffer. The complex was resolved by electro- phoresis on a 5-20% SuperSep gel (Wako) in 0.5× TBE buffer at 4°C, transferred to N+nylon membrane, and detected by streptavidin-HRP using a Lightshift chemilu- minescence electrophoretic mobility shift assay (EMSA) kit (PIERCE). Super-shift reactions were run as described above with the exception that 2 μg of polyclo- nal anti-NF- B-p50 and -p52 antibodies were used. Assays of Tenascin-C levels in conditioned medium by means of ELISA The levels of high molecular-weight TN-C protein were determined using an ELISA kit (IBL) for the conditioned medium of MKN45/mock and MKN45/PAR1 at 3, 6, and 12 hr after the addition of 15 nM a-thrombin. The collected samples were concentrated by using VIVAspin (Vivascience, Stonehouse, UK) ultra-filtration units and incubated in 96-well ELISA plates for 1 hr at 37°C. After washing out unreacted antibody, HRP-conjugated anti-TN-C was added, followed by incubation for 30 min at 4°C, and the color intensity was determined at 450 nm. Results were calculated from the mean absorbance of duplicate wells. Assays of TN-C initiated phosphorylation of EGFR MKN45/PAR1 was separately exposed to 15 nM a-thrombin for either 3 hr or 12 hr period. We then collected each separate conditioned medium and filtered it removing proteins with molecular weights lower than 200 kDa, including a-thrombin, and retained the high molecular weight protein concentrate. The concentrate then underwent two rounds consisting of a ten-fold dilution with PBS followed by filtration to isolate the same high molecular weights proteins. The level of a- thrombin in the resultant concentrates was estimated by means of SDS-PAGE and Western blot and was found