Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Page 7 of 14 Figure 6 Proliferation assay in MKN28. The effect of a-thrombin stimulation on cellular features of MKN28 cells intrinsically expressing PAR1. This figure shows increases in cell proliferation rate of MKN28 cells when stimulated with 15 nM a-thrombin and the preventative effect of a PAR1-antagonist, SCH79797, on the MKN28 cell proliferation. The assay was carried out according to the same procedure as that described in Figure 2 legend. both MKN45/PAR1 and MKN28 was activated by a-thrombin, resulting in cell proliferation and invasion. PAR1 activation induces NF- B activation Having established the ability of PAR1 activation to sti- mulate both proliferation and invasion of MKN45/ PAR1, we next evaluated the ability of PAR1 activation to stimulate transcriptional events. The EMSA showed that the PAR1 agonist a-thrombin can induce the acti- vation of NF- B within 30 min of treatment of the MKN45/PAR1. The EMSA signal persisted for 24 hr (Figure 8). MKN45/mock and MKN45/PAR1 not exposed to a-thrombin did not present any retarded EMSA bands (Figure 8). We performed a super-shift Figure 7 Invasion assay in MKN28. This shows elevated invasion functions of MKN28 cells caused by thrombin stimulation and the concentration-dependent prevention effect of a PAR1-antagonist, SCH79797. (Date were expressed as mean values ± SD from triplicate experiments.)