Journal: International journal of oncology

Article Title: Thrombin conducts epithelial‑mesenchymal transition via protease‑activated receptor‑1 in human gastric cancer.

doi: 10.3892/ijo.2014.2651

Figure Lengend Snippet: Figure 1. Protease‑activated receptor‑1 (PAR1) activation-dependent expression of epithelial‑mesenchymal transition (EMT) markers in MKN45/PAR1 and MKN74 cells. Whole‑cell and nuclear‑cell lysates of MKN45/mock, MKN45/PAR1 and MKN74 cells were probed for EMT‑marker expression by western blotting. (A) EMT‑marker expression remains the same in MKN45/mock cells treated with α‑thrombin or α‑thrombin plus SCH79797 for 24 h. (B) MKN45/PAR1 and MKN74 cells treated with α‑thrombin present decreased levels of E‑cadherin and β‑catenin and increased levels of fibronectin and vimentin in whole‑cell lysates over time. Nuclear lysates of these α‑thrombin‑treated cells presented an increase in β‑catenin over time suggesting movement of β‑catenin into the nucleus. (C) When treated with α‑thrombin and SCH79797, MKN45/PAR1 and MKN74 cells present results similar to MKN45/mock cells. Equal loading of protein was confirmed with anti‑GAPDH antibody.

Article Snippet: The selective PAR1 antagonist Sch79797 (catalog no. 1592) (ic50=70 nM) was purchased from tocris Bioscience (avonmouth, uK) (25).

Techniques: Activation Assay, Expressing, Western Blot

Journal: International journal of oncology

Article Title: Thrombin conducts epithelial‑mesenchymal transition via protease‑activated receptor‑1 in human gastric cancer.

doi: 10.3892/ijo.2014.2651

Figure Lengend Snippet: Figure 2. Fluorescence immunocytochemical staining of fibronectin and E‑cadherin in MKN45/mock, MKN45/PAR1 and MKN74 cells, when treated with α‑thrombin or α‑thrombin plus SCH79797. (A) MKN45/mock cells present no significant changes in fibronectin and E‑cadherin expression. (B) MKN45/APR1 and MKN74 cells treated with α‑thrombin presented a decreased level of E‑cadherin expression and an enhanced level of fibronectin expression. (C) MKN45/APR1 and MKN74 cells treated with α‑thrombin and SCH79797, presented fibronectin and E‑cadherin expression levels similar to that of untreated cultures of these cells.

Article Snippet: The selective PAR1 antagonist Sch79797 (catalog no. 1592) (ic50=70 nM) was purchased from tocris Bioscience (avonmouth, uK) (25).

Techniques: Fluorescence, Staining, Expressing

Journal: International journal of oncology

Article Title: Thrombin conducts epithelial‑mesenchymal transition via protease‑activated receptor‑1 in human gastric cancer.

doi: 10.3892/ijo.2014.2651

Figure Lengend Snippet: Figure 3. Electrophoretic mobility shift assays (EMSAs), demonstrate specific interaction with the E‑cadherin promoter. (A) EMSAs were performed with nuclear extracts from MKN45/mock and MKN74 cells and demonstrate an E‑cadherin E‑box 1‑3 nuclear protein complex. No levels of specific E‑box complexes are seen in MKN45/mock and MKN74 cells. These cells are the control. (B) These lanes show nuclear extracts from MKN45/PAR1 and MKN74 cells, treated with α‑thrombin for 12 h, and demonstrate an E‑cadherin E‑box1‑3 nuclear protein complex. Higher levels of specific E‑box complexes are seen in MKN45/PAR1 and MKN74 cells treated with α‑thrombin that overexpress relative to control cells. (C) Specific complexes were inhibited by protease‑activated receptor‑1 (PAR1) selective antagonist SCH79797.

Article Snippet: The selective PAR1 antagonist Sch79797 (catalog no. 1592) (ic50=70 nM) was purchased from tocris Bioscience (avonmouth, uK) (25).

Techniques: Electrophoretic Mobility Shift Assay, Control

Journal: International journal of oncology

Article Title: Thrombin conducts epithelial‑mesenchymal transition via protease‑activated receptor‑1 in human gastric cancer.

doi: 10.3892/ijo.2014.2651

Figure Lengend Snippet: Figure 4. Snail detected in nuclear lysate. The impact of α‑thrombin treatment of MKN45/PAR1 and MKN74 cells upon nuclear localization of transcription factors in these cells (Twist, Snail and E12/E47) was profiled by means of western blotting. Twist and E12/E47 were not able to migrate into the nucleus, when treated with α‑thrombin while Snail was able to do so, when these cells were treated with α‑thrombin for 12 h.

Article Snippet: The selective PAR1 antagonist Sch79797 (catalog no. 1592) (ic50=70 nM) was purchased from tocris Bioscience (avonmouth, uK) (25).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

doi: 10.1038/s41598-018-29669-z

Figure Lengend Snippet: mIVD expression of PAR1 thrombin receptors. ( a ) Tissue was obtained from C57BL/6 mice for RNA and protein extraction. Quantitative PCR was performed using specific primers for Par1 and Hprt . The ratio of each gene compared with that of Hprt was calculated, and the value of 1 was assigned to brain tissue. ( b ) Western blotting analysis showed that PAR1 was sufficiently expressed in organ tissues including NP, AF, and total mIVD. NCs were loaded for the negative control and murine lung protein was loaded as a positive control. GAPDH was used as an internal control. ( c ) Immunohistological analysis of PAR1 (brown) expression in NP, AF and CEP (right) at high magnification and in whole mIVDs (left) at low magnification. No positive cell staining was observed in the control Ig-treated sections. Lung tissues were stained with anti-PAR1 Ab for the positive control. Representative images from 3 independent experiments are shown (arrow, positive cell; scale bar, 200 or 10 μm). Abbreviations: AF, annulus fibrosis; CEP, cartilage endplate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mIVD, murine intervertebral disc; NCs, no cells; NP, nucleus pulposus; TF, tissue factor. See Supplementary Fig. for examples of uncropped images for each antibody.

Article Snippet: Purchased for use in this study was a PAR1 antagonist (YFLLRNP) from AnaSpec, Inc. (OH, Fremont, CA, USA), PAR1 inhibitor (SCH79797) from Santa Cruz Biotechnology, Inc. (TX, USA), a PI3K inhibitor (LY294002) obtained from Cayman Chemical (Ann Arbor, MI, USA), a MAPK-ERK inhibitor (PD98059), a P38 inhibitor (SB203580) from Merck KGaA (Darmstadt, Germany), and mouse MCP-1 Ab from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Negative Control, Positive Control, Control, Staining

Journal: Scientific Reports

Article Title: Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

doi: 10.1038/s41598-018-29669-z

Figure Lengend Snippet: Induction of MCP-1 in mIVD via thrombin/PAR1 signaling. ( a ) mIVDs were cultured in the presence or absence of 100 nM of thrombin for 72 hours. The culture supernatants were collected and subjected to a cytokine protein array. The table indicates the corresponding cytokines on the protein array membrane. ( b ) mIVDs were stimulated with thrombin and quantitative PCR was performed using specific primers for Mcp-1 and Hprt . The ratio of each gene to that of Hprt was calculated, and the value of 1 was assigned to no treatment. ( c , d ) mIVDs were stimulated with thrombin and culture supernatants were collected to measure the concentration of MCP-1 using an ELISA assay. ( e , g ) mIVD were stimulated with thrombin (100 nM) with or without a PAR1 inhibitor (1 µg/mL) for 72 hours. The cell lysates and supernatants were subjected to Western blotting analysis with anti-MCP-1 and anti-GAPDH Ab or analyzed using the ELISA system. ( f ) Images of panel (e) were captured using an LAS-4000 camera system and quantified by imageJ software. Values represent the mean ± SD. * p < 0.05 compared with the corresponding control. Similar results were obtained in at least 3 independent experiments. Abbreviations: C, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Th, thrombin. See Supplementary Fig. for examples of uncropped images for each antibody. See Supplementary Fig. for additional experiments that are the same as Fig. 3e–g with another PAR1 inhibitor. (0.3 µM).

Article Snippet: Purchased for use in this study was a PAR1 antagonist (YFLLRNP) from AnaSpec, Inc. (OH, Fremont, CA, USA), PAR1 inhibitor (SCH79797) from Santa Cruz Biotechnology, Inc. (TX, USA), a PI3K inhibitor (LY294002) obtained from Cayman Chemical (Ann Arbor, MI, USA), a MAPK-ERK inhibitor (PD98059), a P38 inhibitor (SB203580) from Merck KGaA (Darmstadt, Germany), and mouse MCP-1 Ab from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Protein Array, Membrane, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Software, Control

Journal: Scientific Reports

Article Title: Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

doi: 10.1038/s41598-018-29669-z

Figure Lengend Snippet: MCP-1 produced in mIVDs induced macrophage migration. ( a – f ) mIVDs were cultured in the presence or absence of 100 nM of thrombin and with or without anti-MCP-1 neutralizing Ab (2 µg/mL) or PAR1 inhibitor (1 µg/mL) for 72 h. The culture supernatants were collected and poured to the lower Chemotaxicell chamber. Upper wells were populated with 3 × 10 5 macrophages in 500 µL of DMEM containing 0.1% FBS. After incubation for 6 hours at 37 °C, cells that had migrated to the lower surface were fixed and stained with crystal violet (scale bar, 50 μm). ( b , d , f ) The cells on the lower chamber surface were counted in 8–10 fields under high-power magnification (200×). TNF-α (10 ng/mL) treatment was used as positive control. * p < 0.05 compared with the corresponding control. Similar results were obtained in at least 3 independent experiments. Abbreviations: C, control; Th, thrombin.

Article Snippet: Purchased for use in this study was a PAR1 antagonist (YFLLRNP) from AnaSpec, Inc. (OH, Fremont, CA, USA), PAR1 inhibitor (SCH79797) from Santa Cruz Biotechnology, Inc. (TX, USA), a PI3K inhibitor (LY294002) obtained from Cayman Chemical (Ann Arbor, MI, USA), a MAPK-ERK inhibitor (PD98059), a P38 inhibitor (SB203580) from Merck KGaA (Darmstadt, Germany), and mouse MCP-1 Ab from R&D Systems (Minneapolis, MN, USA).

Techniques: Produced, Migration, Cell Culture, Incubation, Staining, Positive Control, Control

Journal: Scientific Reports

Article Title: Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

doi: 10.1038/s41598-018-29669-z

Figure Lengend Snippet: Induction of MCP-1 in mIVDs via thrombin/PAR1 signaling. ( a ) mIVDs were stimulated with thrombin (100 nM) with or without the PI3K inhibitor LY294002 (1 µM) and the inhibitor of MAPK-ERK, PD98059 (1 µM), for 72 hours. The supernatants were analyzed using an ELISA system. ( b , c ) mIVDs were stimulated with thrombin in the presence or absence of LY294002 and PD98059. The cell lysates were subjected to Western blotting analysis with Abs specific for phosphorylated AKT, AKT, phosphorylated ERK P42/44, and ERK P42/44. Values represent the mean ± SD. * p < 0.05 compared with the corresponding control. Similar results were obtained in at least 3 independent experiments. Abbreviations: C, control; LY, LY294002; PD, PD98059; Th, thrombin. See Supplementary Figs – showing uncropped images for each antibody.

Article Snippet: Purchased for use in this study was a PAR1 antagonist (YFLLRNP) from AnaSpec, Inc. (OH, Fremont, CA, USA), PAR1 inhibitor (SCH79797) from Santa Cruz Biotechnology, Inc. (TX, USA), a PI3K inhibitor (LY294002) obtained from Cayman Chemical (Ann Arbor, MI, USA), a MAPK-ERK inhibitor (PD98059), a P38 inhibitor (SB203580) from Merck KGaA (Darmstadt, Germany), and mouse MCP-1 Ab from R&D Systems (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

doi: 10.1038/s41598-018-29669-z

Figure Lengend Snippet: Induction of MMP-3 in mIVDs via thrombin/PAR1 signaling. ( a , c ) mIVDs were stimulated with thrombin (100 nM) with or without a PAR1 inhibitor (1 µg/mL) for 72 hours. The cell lysates and supernatants were subjected to Western blotting analysis with anti-MMP-3 and anti-GAPDH Abs or analyzed using the ELISA system. GAPDH was loaded as a control. ( b ) Images of Fig. 6a were captured using an LAS-4000 camera system and quantified by imageJ software. Values represent the mean ± SD. * p < 0.05 compared with the corresponding control. Similar results were obtained in at least 3 independent experiments. ( d ) mIVDs were stimulated with thrombin with or without PAR1 inhibitor for 72 hours. Immunohistological analyses were performed for MMP-3 (brown) expression in NP, AF, and CEPs (right) at high magnification and in whole mIVDs (left) at low magnification. No positive cell staining was observed in the untreated and control sections. TNF-α-treated tissues were stained with anti-MMP-3 Ab as a positive control. Representative images from 3 independent experiments are shown (arrow, positive cell; scale bar, 200 or 10 μm). Abbreviations: AF, annulus fibrosis; C, control; CEP, cartilage endplate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mIVD, murine intervertebral disc; NP, nucleus pulposus; NT, not treated; Th, thrombin. See Supplementary Fig. for examples of uncropped images for each antibody.

Article Snippet: Purchased for use in this study was a PAR1 antagonist (YFLLRNP) from AnaSpec, Inc. (OH, Fremont, CA, USA), PAR1 inhibitor (SCH79797) from Santa Cruz Biotechnology, Inc. (TX, USA), a PI3K inhibitor (LY294002) obtained from Cayman Chemical (Ann Arbor, MI, USA), a MAPK-ERK inhibitor (PD98059), a P38 inhibitor (SB203580) from Merck KGaA (Darmstadt, Germany), and mouse MCP-1 Ab from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control, Software, Expressing, Staining, Positive Control

Journal: Scientific Reports

Article Title: Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

doi: 10.1038/s41598-018-29669-z

Figure Lengend Snippet: Treatment of mIVDs with thrombin induced disc degeneration. mIVDs were stimulated with thrombin (100 nM) in the absence or presence of a PAR1 inhibitor (1 µg/mL) for 72 hours. Safranin-O and fast green stains were performed for proteoglycan expression (orange) in NP, AF, and CEPs at high magnification (right) and in whole mIVDs (left) at low magnification. TNF-α-treated tissues were stained with Safranin-O and fast green as a positive control for mIVD degeneration. Representative images from 3 independent experiments are shown (scale bar, 200 or 10 μm). Abbreviations: AF, annulus fibrosis; C, control; CEP, cartilage endplate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mIVD, murine intervertebral disc; NP, nucleus pulposus; NT, not treated; Th, thrombin.

Article Snippet: Purchased for use in this study was a PAR1 antagonist (YFLLRNP) from AnaSpec, Inc. (OH, Fremont, CA, USA), PAR1 inhibitor (SCH79797) from Santa Cruz Biotechnology, Inc. (TX, USA), a PI3K inhibitor (LY294002) obtained from Cayman Chemical (Ann Arbor, MI, USA), a MAPK-ERK inhibitor (PD98059), a P38 inhibitor (SB203580) from Merck KGaA (Darmstadt, Germany), and mouse MCP-1 Ab from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Staining, Positive Control, Control

Journal: The American journal of pathology

Article Title: Protein C concentrate controls leukocyte recruitment during inflammation and improves survival during endotoxemia after efficient in vivo activation.

doi: 10.1016/j.ajpath.2011.07.023

Figure Lengend Snippet: Figure 8. Role of thrombomodulin (TM), endothelial protein C receptor (EPCR), and protease-activated receptor-1 (PAR1) for protein C (PC)-induced inhibition of leukocyte adhesion during tumor necrosis factor- (TNF-)- induced inflammation. Leukocyte adhesion in 3 hours of TNF--stimulated cremaster muscle venules of TM mutant (TM pro/pro) mice with and without administration of 100 U/kg PC for 3 hours before observation were compared to wild-type (WT) control mice (A). Furthermore, PC-treated (100 U/kg, 3 hours) mice were pretreated with the EPCR-blocking antibody RCR252 or PAR-1 inhibitor SCH79797 to observe leukocyte adhesion in TNF--stimu- lated cremaster muscle venules and compared with respective controls (B). All values were generated from at least three mice per group and are presented in adherent leukocytes per mm2 as mean SEM. Significant differences (*P 0.05) to control are indicated.

Article Snippet: In certain experiments, the EPCR antibody RCR252 (30 g/mouse; Abcam, Cambridge, UK) directed to the PC binding domain of EPCR or the selective PAR-1 Inhibitor SCH79797 (44 g/mouse, Tocris Bioscience, Bristol, UK34) was injected into the tail vein 5 minutes before PC application.

Techniques: Inhibition, Mutagenesis, Control, Blocking Assay, Generated