Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: The expression of PAR1 mRNA and protein in gastric cancer cell lines . (A) The expression of PAR1 mRNA in 7 gastric cancer cell lines by RT-PCR. PAR1 mRNA was confirmed in MKN28, MKN74 and NUGC3. (B) Parent MKN45 cells and MKN45 cells transfected with pcDNA3.1-empty-vector alone (designated as MKN45/mock) have no detectable expression of PAR1 protein, and MKN28 and MKN45 cells transfected with PAR1 cDNA bearing pCDNA3.1 vector, which was treated 750 μg/ml G418 to select permanent PAR1 expressing clone, (designated as MKN45/PAR1) was confirmed to continuously express high level PAR1 protein by immunoblotting (upper panel). The bottom panel showed a protein band of a house keeping gene, GAPDH as a control. (The final dilution of PAR1 antibody was 1:500, GAPDH was 1:2000.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Control

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Proliferation assay in MKN45/PAR1 with α-thrombin stimulation . MKN45/PAR1 (1.0×10 4 cells/well) 96 hr after adding 15 nM α-thrombin had significantly increased the number. SCH79797, selective PAR1 antagonist, significantly reversed the growth rate of MKN45/PAR1 at 35 and 70 nM and completely abolished thrombin dependent growth progression at 150 nM in the presence of 15 nM α-thrombin.

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Proliferation Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Proliferation assay in MKN45/Mock . This figure showed that the MKN45/mock (1.0×10 4 cells/well) 96 hr after adding 15 nM α-thrombin or 30 μM TFLLRN-NH 2 , specific PAR1 agonist peptide, had not significantly increased the number.

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Proliferation Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Proliferation assay in MKN45/PAR1 with TFLLR-NH 2 . In the same way, MKN45/PAR1 adding 30 μM TFLLR-NH 2 had significantly increased the number, too. And SCH79797 siginificantly reversed the growth rate of MKN45/PAR1 in the presence of 30 μM TFLLR-NH 2 . (Date were expressed as mean values ± SD from triplicate experiments.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Proliferation Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Invasion assay in MKN45/PAR1 . Treatment of MKN45/PAR1 with 15 nM α-thrombin or 30 μM TFLLR-NH 2 resulted in significant enhancement (5 or 6 fold increment relative to untreated control) of its invasion potential. But treatment of MKN45/mock with same conditions of α-thrombin or TFLLR-NH 2 resulted in no significant enhancement. The inhibitory effect of the SCH79797 was clearly observed at 35 and 70 nM. There was the maximum inhibitory effect on the invasion ability of MKN45/PAR1 at 150 nM SCH79797. Basal invading activity was expressed as an absorbance of 560 nm for MKN45/mock cells which were unexposed to α-thrombin. (Date were expressed as mean values ± SD from triplicate experiments.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Invasion Assay, Control, Activity Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Proliferation assay in MKN28 . The effect of α-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 α-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.

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Proliferation Assay, Expressing

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: 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.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Invasion Assay, Concentration Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Activation of NF-κB assessed by EMSA analysis in MKN45/mock and MKN45/PAR1 by α-thrombin stimulation . The gel mobility shift assay was performed on the whole nuclear extract from MKN45/mock and MKN45/PAR1 (in a 12 cm diameter dish) that were incubated in the presence of 15 nM α-thrombin for 0.5, 1, 2, 6, and 24 hr. MKN45/mock samples showed no retardation bands but only intense broad signal due to biotinylated NF-κB cis-element ds-oligo probe on the gel. An arrow on left side of the middle figure indicates the NF-κB/DNA binding complex. In the super-shift assay, antibodies (2 μg) raised against NF-κBp50 and NF-κBp52 were mixed with nuclear extract obtained from PAR 1 -transfectanted cells treated with 15 nMα-thrombin for 0.5 hr. A super-shifted band was clearly detected with NF-κBp50 antibody as marked by an arrowhead, but not detected with NF-κBp52 antibody.

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Activation Assay, Mobility Shift, Incubation, Binding Assay, Super-Shift Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: The mRNA expression of NF-κB target genes by quantitative RT-PCR . We selected 10 target genes of NF-κB that are concerned with cell growth and invasion. Most of the mRNA expression were increased after 15 nM α-thrombin stimulation. The mRNA expression levels of TN-C, Bcl-2, cIAP1 and EGFR reached more than 100 times the relative levels of the control values. Especially, TN-C mRNA expression level was increased. The kinetics of mRNA expression following α-thrombin stimulation in MKN45/PAR1 are different depending upon type of genes tested. The bar height indicates the calculated fold activation values for each time after stimulation with 15 nM α-thrombin. It was based on the condition of no α-thrombin stimulation. (Date were expressed as mean values ± SD from triplicate experiments.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Expressing, Quantitative RT-PCR, Control, Activation Assay

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Detection of the proteins, which were high expression mRNA under α-thrombin, by immunoblotting . MKN45/PAR1 were stimulated with 15 nM α-thrombin for 3, 6, and 12 hr and harvested for preparing whole cell lysates. The clear extracts containing 100 μg protein was developed on SDS-PAGE. The expression of TN-C, Bcl-2, cIAP1, EGFR and GAPDH protein, which was a house keeping gene product as a control, were detected by Western blotting using specific antibodies against respective proteins (The final dilutions of TN-C, Bcl-2, cIAP1 and EGFR antibodies were 1:500, GAPDH was 1:2000). The MKN45/PAR1 were harvested at 3, 6 and 12 hr after 15 nM α-thrombin stimulation and whole cells were lysed using LIPA buffer. Significant EGFR protein expression was detected in MKN45/mock and unstimulated MKN45/PAR1. The expression above basal level was clearly observed for TN-C, Bcl-2, cIAP1 and EGFR proteins 6 hr after addition of 15 nM α-thrombin.

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Expressing, Western Blot, SDS Page, Control

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Effect of NF-κB inhibitor CAPE and a PAR1 antagonist SCH79797 on trans-criptional activation by thrombin stimulation of NF-κB target genes . NF-κB inhibitor or PAR1 antagonist was added at 25 μg/ml or 70 nM, respectively, to the culture medium with 15 nM α-thrombin. Six hour later, the treated cells were harvested for preparing total RNA, which was subjected to quantification for mRNA expression of four NF-κB target genes, such as, TN-C, Bcl-2, cIAP1 and EGFR by real time RT-qPCR. Each mRNA expression level after 6 hr thrombin stimulation is 100%, the suppressive effect of inhibitor or antagonist was expressed as % inhibition. (Date were expressed as mean values ± SD from triplicate experiments.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Inhibition

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: TN-C concentration determined by ELISA in the conditioned medium of α-thrombin stimulated MKN45/PAR1 . Six cm diameter dishes with 50% confluency of MKN45/mock, MKN45/PAR1 were used for preparing the conditioned medium. α-thrombin was added at 15 nM to 6 dishes with MKN45/PAR1, and the conditioned medium was collected at indicated time (3, 6, and 12 hr) and cleared supernatant by centrifugation was subjected to ELISA assay which can detect only high molecular TN-C. The concentration of high molecular TN-C in the conditioned medium was expressed as μg/ml. (Date were expressed as mean values ± SD from triplicate experiments.)

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Centrifugation

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Increases in EGFR protein expression and phosphorylation of EGFR at Tyr1173 in MKN45/PAR1 stimulated with α-thrombin . MKN45/PAR1 were stimulated with 15 nM α-thrombin for 1, 3, 6, and 12 hr and harvested for preparing whole cell lysates. The clear extracts containing 100 μg protein was developed on SDS-PAGE. We detected by western blotting experiment using an antibody raised against total EGFR (dilution of 1: 500) was increased 3 hr after addition of 15 nM α-thrombin to MKN45/PAR1. But we detected using an antibody raised against phosphotyrosine (Tyr1173) that tyrosine phosphorylation of EGFR (dilution of 1:500) was slightly increased 1 hr after addition of 15 nM α-thrombin, and following 3 hr, phospho-EGFR was dramatically increased.

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Expressing, Phospho-proteomics, SDS Page, Western Blot

Journal: BMC Cancer

Article Title: The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

doi: 10.1186/1471-2407-10-443

Figure Lengend Snippet: Detection of phosholyrated EGFR by TN-C . The conditioned medium of PAR1/MKN45 culture pre-stimulation by 15 nM α-thrombin for 3 and 12 hr was collected and partially purified by repeated concentration using ultra-filtration spin columns able to cutoff macromolecules below 200 kDa. MKN45/PAR1 were incubated either with the concentrated conditioned medium for 6 hr, which were pre-stimulated for 3 hr or 12 hr by 15 nM α-thrombin. These whole lysates of MKN45/PAR1 treated with the conditioned medium pre-stimulated for 12 hr by 15 nM α-thrombin was detected remarkably enhanced tyrosine phospholyration of the EGFR, but these whole lysates of MKN45/PAR1 with the conditioned medium pre-stimulated for 3 hr by 15 nM α-thrombin was not detected the tyrosine phospholyration of the EGFR (left). The whole lysates of MKN45/PAR1 treated with the concentrated conditioned medium with TN-C-neutralizing antibody (25 μg/ml), which was pre-stimulated for 12 hr by 15 nM α-thrombin, show no-increased in phosphor-EGFR protein quantity (right).

Article Snippet: The selective PAR1 antagonist SCH79797 (catalog #1592) (IC50 = 70 nM) and PAR1 agonist TFLLR-NH 2 (catalog #1464) were purchased from Tocris Bioscience (Anonmouth, UK) [ ].

Techniques: Purification, Concentration Assay, Filtration, Incubation

Journal: Hippocampus

Article Title: Septohippocampal pathways contribute to system consolidation of a spatial memory: sequential implication of GABAergic and cholinergic neurons.

doi: 10.1002/hipo.20837

Figure Lengend Snippet: FIGURE 4. Representative images of ChAT (top) and Parv (bottom) immunostainings in SHAM (A and E), SAP (B and F), OREX (C and G), and SAP/OREX (D and G) rats. For anti-Parv immunostaining, a slice showing nonspecific staining (no primary antibody used during incubation) is also shown. In the MS/vDBB regions (plate modified from Paxinos and Watson, 1998), the two squares on each side delimit the regions where ODs for anti-Parv immunostaning were measured. Cholinergic neurons were mark- edly depleted in SAP (C) and SAP/OREX (D) and relatively spared in OREX (B) rats. GABA neurons were markedly depleted in OREX (G) and SAP/OREX (H) and relatively preserved in SAP (F) rats. Both neuronal populations were unaltered in SHAM animals (A and E).

Article Snippet: As primary antibodies, we used a goat polyclonal antibody directed against ChAT (1:500; Chemicon International, Temecula, CA) and a mouse monoclonal antibody directed against Parv (1:4000; Sigma-Aldrich, P 3088, St. Louis, MO), whereas donkey anti-goat (ChAT) and horse anti-mouse (Parv) biotinylated secondary antibody (1:500, Vector Laboratories International, Burlingame, CA) were used.

Techniques: Immunostaining, Staining, Incubation