Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Bar graph showing effects of PAR1 inhibitors on rat oligodendrocyte precursor cells (OPC) cultured in serum-free defined media. Cell death was measured by LDH release (optical density units; mean ± SEM). At high concentrations both SCH79797 and BMS20061 were toxic to the cells (*p < 0.05).

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Cell Culture

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Bar graph showing effects of plasma alone and in combination with PAR1 inhibitors on rat oligodendrocyte precursor cells (OPC) in culture. Cell death was measured by LDH release (optical density units; mean ± SEM). Plasma at 1/250 concentration added to the defined culture media was toxic to OPC as indicated by increased LDH release. There was no evidence for protection with SCH79797 and BMS20061 at any dose. Control values (no plasma) were significantly less than all groups (*p < 0.05) and there were no statistically significant differences between treatment groups (all comparisons p > 0.5).

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Clinical Proteomics, Concentration Assay, Control

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Cell proliferation in immature mouse brain following periventricular blood injection. A - Ki67 nuclear immunoreactivity (brown stained nuclei; hematoxylin counterstain) is prominent in the frontal periventricular subventricular zone (SVZ, arrow) of control (i.e. no blood injection) mice from all three genotypes (this example is wild type). B – Following blood injection, some of which extends into the frontal horn of the lateral ventricle (arrow), Ki67 immunoreactivity in the SVZ is reduced (this example is a PAR1 knockout). C – Quantitative analysis (ANOVA) shows the proportion of Ki67 immunoreactive cells in the SVZ was significantly reduced in the 24-hour heterozygous (Het; *p = 0.0050) and the 48-hour wild type (WT; *p = 0.0054) mice; the reduction also approached significance in the 48-hour knockout (KO; p = 0.0577) in comparison to controls. Bar = 100 μm.

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Injection, Staining, Control, Knock-Out, Comparison

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Cell death in immature mouse brain following periventricular blood injection. A - Activated caspase 3 immunoreactivity is extremely rare in the SVZ of control mice (brown stained cells; hematoxylin counterstain) (arrow; this example is wild type). B – Following blood injection, activated caspase 3 immunoreactivity is prominent in the striatum (arrow) but very rare in the SVZ (this example is a PAR1 knockout). C – Quantitative analysis shows a statistically significant increase in caspase 3 immunoreactive cells in the striatum of most groups in comparison to intact control (p < 0.007), but there were no significant differences between the blood-injected groups (all p > 0.15; Wilcoxon method). All micrographs were taken at 400× magnification. Bar = 100 μm.

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Injection, Control, Staining, Knock-Out, Comparison

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 mRNA expression

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Microarray

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 is overexpressed in glioma tumor progenitor cells (TPCs) (a), Expression of PAR1 mRNA using real-time PCR, in freshly sorted glioma-derived A2B5+ TPCs (n = 12) relative to normal A2B5+ glial progenitor cells (GPCs) (n = 4) and unsorted cells (UNS) (n = 4), reveals that PAR1 mRNA was significantly upregulated at all stages of glioma development. (b–c) Relative quantification of PAR1 gene (b) and protein expression (c) using RT-PCR (b) and flow cytometry (c) detection in GBM-derived glioma-initiating cell lines (GICLs) established from unsorted or A2B5+ cells (*) maintained in serum-free media (SFM) supplemented with FGF, EGF (20 ng/ml) and PDGF (10 ng/ml) for less than 10 passages; and commercially available adherent GBM cells U87 and U251 (c) cultured in 10% serum culture conditions. Comparable quantities of cDNA were ensured by amplification of GAPDH (b). (d–f) Flow cytometry analysis of PAR1, A2B5 and CD133 expression in GBM-derived GICLs. Representative scatter plot of GICL8 stained with A2B5 (bottom right), PAR1 (upper left), both (upper right), or their corresponding isotype controls (bottom left) (D). Error bars indicate Means ± s.e.m.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cell Culture, Amplification, Staining

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: Analysis of PAR1 expression in relation with TCGA-defined glioblastoma subtypes and copy number variations (a–b), PAR1 gene expression was enriched in the CL subtype of GBM defined by the TCGA (n= 483) relative to normal brain tissue (n = 10) (a) but was not correlated with the glioma-Cpg Island Methylator Phenotype (G-CIMP) subtype. (b, c) Expression value of PAR1 gene expression in low-grade gliomas (n = 468) derived from the TCGA showing a significant enrichment of PAR1 in anaplastic AST, relative to grade II OLG and OA. Black lines in each group indicate mean ± s.e.m. 1 way ANOVA, P < 0.0001; *P < 0.05; **P < 0.01; ***P < 0.001 after Tukey’s multiple test comparison. (d) PAR1 expression was significantly correlated with EGFR, PTEN and CDKN2A copy number variations. AST, astrocytoma; CL, classical; OLG,: oligodendroglioma; OA, oligoastrocytoma; II and III: WHO grades II and III, respectively; MES, mesenchymal; NL, neural; PN: proneural.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Expressing, Gene Expression, Derivative Assay, Comparison

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: Lentiviral induced PAR1 knockdown (KD) validation. Validation of PAR1 gene (a, b, e) and protein (c–e) induced silencing using quantitative RT-PCR (a, b) and flow cytometry (c, d) A2B5+ derived glioma-initiating cell lines (GICLs), 6 days after transduction with two different PAR1 KD lentiviruses, compared with glioma cells transduced with a SCR. Gene expression levels normalized to GAPDH. One-way ANOVA with repeated measures; *P < 0.05; **P < 0.01; ***P < 0.001 after Tukey post-hoc comparisons. Means ± s.e.m.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Knockdown, Biomarker Discovery, Quantitative RT-PCR, Flow Cytometry, Derivative Assay, Transduction, Gene Expression

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 silencing inhibits the growth and self-renewal of glioma TPCs. (a) Representative photomicrograph illustrating the number of A2B5+ TPCs derived from GICL-8 6 days after transduction with either PAR1-KD or control lentiviruses. Scale bar, 100 µm. (b–g) Effects of PAR1 silencing on the in vitro expansion (b, c), proliferation (d, e), survival (f, g) and clonal sphere formation (h, i) of A2B5+ TPCs derived from two glioma-initiating cell lines (GICL8 and GICL9), 6 days after transduction with different PAR1 knockdown (KD) lentiviruses, compared with scrambled lentivirus (SCR) and control (CT) untransduced cells. (b, c) Lentiviral KD of PAR1 significantly reduced the number of A2B5+ GICL relative to both SCR shRNAi-transduced and non-transduced CT cells. (d, e) EdU incorporation in association with propidium iodide (PI) staining revealed that GICLs subjected to PAR1 KD manifested fewer cells in S phase relative to SCR and CT cells. (f, g) PAR1 KD significantly increased the percentage of apoptotic cells as determined by flow cytometry analysis of Annexin V, relative to SCR and CT cells. KD, Knockdown. Means ± s.e.m. One-way ANOVA with repeated measures (P < 0.0001) with Tukey post-hoc comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Post-hoc comparisons between PAR1 KD cells and SCR and non-transduced control (CT) cells are illustrated by orange and black stars, respectively; three independent experiments for each cell line. (h, i) GICLs were plated into a 96-well plates for limiting dilution sphere formation assay, and counted 14 days later; wells were then scored for the presence or absence of sphere growth. The log fraction of the negative wells (non-responding) was plotted as a function of cell density per well.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Derivative Assay, Transduction, Control, In Vitro, Knockdown, Staining, Flow Cytometry, Tube Formation Assay

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: PAR1 silencing suppresses the in vivo growth of TPC-derived tumors and prolongs survival Effects of PAR1 silencing on the in vivo expansion of A2B5+ GICLs derived from two different GBM (28 000 cells per animal, n = 5–6 mice per group), 4 weeks after transduction with PAR1 knockdown (KD) lentiviruses, compared with scrambled lentivirus (SCR) and control (CT) untransduced cells. (a) Hematoxylin-eosin stained sections of xenografts following intracranial implantation of A2B5+ TPCs. (b–g) Graphs representing the stereological analysis of the tumor extension, measured along the antero-posterior axis of xenograft mice brain (b, c); tumor volume (d, e); and proliferation of glioma TPCs as shown by the number of xenografted cells stained with the anti-human nuclei antigen (HNA) co-expressing the mitotic marker Ki67 (f, g), demonstrating a prominent inhibitory effect of PAR1 silencing on the tumorigenicity and mitotic activity of glioma A2B5+ TPCs relative to SCR and CT cells. Means ± s.e.m. P-values calculated using one-way ANOVA (P < 0.0001) followed by Tukey post-hoc comparisons with *P < 0.05; **P < 0.01; ***P < 0.001. Post-hoc comparisons between cells transduced with PAR1 KD shRNAi, compared with those transduced with SCR shRNAi untransduced CT cells are illustrated by orange and black stars, respectively. (h–i) Kaplan–Meier curves show an increase in median survival of mice bearing intracranial glioma TPCs transduced with PAR1 shRNA relative to SCR and CT mice. Log-rank analysis, (P < 0.0001) followed by pairwise comparison between all groups with **P < 0.01 and ***P < 0.001.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: In Vivo, Derivative Assay, Transduction, Knockdown, Control, Staining, Expressing, Marker, Activity Assay, shRNA, Comparison

Journal: Oncogene

Article Title: PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

doi: 10.1038/onc.2015.452

Figure Lengend Snippet: Pharmacological inhibition of PAR1 impedes glioma TPC expansion and migration in vitro. Effects of the specific PAR1 inhibitors SCH79797 and SCH530348 on the in vitro expansion (a–d) and migration (e–h) of A2B5+ GICL derived from GBMs. Dose-dependent growth of GBM-derived TPCs was measured by counting the number of cells 4 days after administration of SCH79797 (a, b) and SCH530348 (c, d) relative to highest concentration of vehicle control (DMSO). P-values were calculated using one-way ANOVA with repeated measures followed by Tukey post-hoc comparisons with *P < 0.05; **P < 0.01; ***P < 0.001. Results were obtained from three independent experiments for each cell line. Migration was measured by assessing the average cell speed of A2B5+ GICLs in a dose-dependent manner, 24 h after administration of SCH79797 (e, f) and SCH530348 (g, h), relative to 1% DMSO vehicle control solution. P-values were calculated using Kruskal–Wallis test followed by Dunn’s multiple comparison test post-hoc comparisons with **P < 0.01; ***P < 0.001. Means ± s.e.m.

Article Snippet: The selective PAR1 antagonists SCH 79797 and SCH 530348 were purchased, respectively from Tocris Bioscience (Ellisville, MO, USA) and Axon medchem (Axon 1755) and dissolved in DMSO as per the manufacturer’s instructions.

Techniques: Inhibition, Migration, In Vitro, Derivative Assay, Concentration Assay, Control, Comparison

Journal: Experimental & Molecular Medicine

Article Title: Distribution and impact of p16 INK4A+ senescent cells in elderly tissues: a focus on senescent immune cell and epithelial dysfunction

doi: 10.1038/s12276-024-01354-4

Figure Lengend Snippet: a The schematic image depicts the effect of senescent immune cells on the surrounding microenvironment of tissues. b A dot plot illustrates the incoming and outgoing strength (interaction count) in each cell type in GSE178341. c The ingoing and outgoing interaction strength across 18 different signaling pathways in each cell type. A blue box indicates the interaction strength of p16 INK4A- T cells, while a red box indicates the interaction strength of p16 INK4A+ T cells. The y axis of the top bar graph indicates the average number of interactions or connections for each cell type within the signaling network. d The interaction strength of PARs signaling network is displayed. e The strength of sender, receiver, mediator, and influencer in the PARs signaling pathway network were examined in each cell type. f The IHC analysis of PAR1 (left panel) and PAR2 (right panel) in normal colon tissues from young and elderly individuals is shown . g The violin plot displays the mRNA expression level of GzmA in p16 INK4A- and p16 INK4A+ T cells from GSE178341 (left panel). The violin plot illustrates the mRNA expression of GzmA in T cells from young and old individuals (right panel). h The IHC analysis of GzmA was performed in colon tissues from young and old individuals, respectively (left panel). The right panel shows the quantification data. The data is presented as mean ± standard deviation. “Young” and “Old” indicate the young and the elderly individuals, respectively. The p -value is calculated using Mann–Whitney U test. i The multiplex IHC analysis shows the expression of CD3 (brown) and GzmA (red) (upper panel) and p16 INK4A (brown) and GzmA (red) (lower panel) in old individuals, respectively. j The multiplex IHC analysis shows the expression of GzmA (brown) and PAR1 (red) (upper panel) and GzmA (brown) and PAR2 (green) (lower panel) in old individuals, respectively.

Article Snippet: Human colon epithelial cells were pre-treated with PAR1 antagonist (100 nM, SCH79797, HY-14993, MedChemExpress, Monmouth Junction, NJ) or PAR2 antagonist (20 μM, AZ3451, HY-112558, MedChemExpress) for 1 h and incubated with recombinant human granzyme A protein (50 ng/ml, ab157288, Abcam) for 1day.

Techniques: Protein-Protein interactions, Expressing, Standard Deviation, MANN-WHITNEY, Multiplex Assay

Journal: Frontiers in Neuroscience

Article Title: Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

doi: 10.3389/fnins.2012.00133

Figure Lengend Snippet: PAR-APs and thrombin activate cultured myenteric neurons . Representative traces of voltage sensitive dye recordings showing neuronal responses to a 2 s spritz application (indicated by the horizontal gray bar) of PAR1-AP, PAR2-AP, PAR4-AP, and thrombin. Recordings were made in four 2 s long recording periods with 5–6 s intervals in between (indicated by the symbol between the traces). Every peak represents an action potential. (A) Representative traces from cultured human myenteric neurons to human specific PAR-APs and thrombin show comparable responses to PAR1-AP and thrombin but no response to PAR2-AP and a minor response to PAR4-AP. (B) Guinea pig cultured myenteric neurons fire action potentials in response to PAR1-AP, PAR2-AP, PAR4-AP, and thrombin; the PAR4 response is rather moderate.

Article Snippet: The PAR1 antagonist SCH79797 (N 3 -cyclopropyl-7-[[4-(1-methylethyl)phenyl] methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine dihydrochloride; Biozol Diagnostica, Eching, Germany) was dissolved in waterfree 100% dimethyl sulfoxide (Acros Organics, Geel, Belgium; Ahn et al., ).

Techniques: Cell Culture

Journal: Frontiers in Neuroscience

Article Title: Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

doi: 10.3389/fnins.2012.00133

Figure Lengend Snippet: Analysis of neural actions of PAR-APs and thrombin in cultured human and guinea pig myenteric neurons . The graphs represent (from top to bottom) the proportion of neurons per cluster responding to a specific PAR activator, the specific PAR activator evoked spike frequency and the neuroindex which is the product of spike frequency and proportion of responding neurons. Data are illustrated with scatter plots showing the 25%/75% and the bars indicating the 10%/90% percentiles. PAR1-AP induced the strongest effect in human myenteric neurons, whereas PAR4-AP evoked only weak responses and PAR2-AP no response. Thrombin also activated human myenteric neurons but to a lesser degree than PAR1-AP. The PAR1-AP effect in human neurons is blocked by the specific PAR1 antagonist SCH79797. In guinea pig myenteric neurons the responsiveness to PAR1-AP, PAR2-AP, and thrombin was similar, but PAR4-AP evoked a spike discharge in less neurons at a significantly lower frequency. *Indicates significant differences to PAR1-AP in human neurons; # indicates significant differences to PAR1-AP in guinea pig neurons. Numbers in parenthesis indicate numbers of tissue/clusters/neurons.

Article Snippet: The PAR1 antagonist SCH79797 (N 3 -cyclopropyl-7-[[4-(1-methylethyl)phenyl] methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine dihydrochloride; Biozol Diagnostica, Eching, Germany) was dissolved in waterfree 100% dimethyl sulfoxide (Acros Organics, Geel, Belgium; Ahn et al., ).

Techniques: Cell Culture

Journal: Frontiers in Neuroscience

Article Title: Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

doi: 10.3389/fnins.2012.00133

Figure Lengend Snippet: Response pattern to PAR-APs in guinea pig cultured myenteric neurons demonstrates receptor clustering . (A) Image shows outlines of a cluster of guinea pig cultured myenteric neurons stained with the voltage sensitive dye Di-8-ANEPPS. (B) Representative traces of a neuron [marked with a star in (A) ] that responded to PAR1-AP and PAR2-AP (2 s spritz application indicated by a gray horizontal bar). Recordings were made in four 2 s long recording periods with 5–6 s intervals in between (indicated by the symbol between the traces). Every peak represents an action potential. (C) Pairwise application of PAR-APs revealed functional coexpression patterns. Proportions of neurons responding to two PAR-APs are expressed relative to the proportion of neurons responding to any PAR-AP in that particular set of experiment (100%).

Article Snippet: The PAR1 antagonist SCH79797 (N 3 -cyclopropyl-7-[[4-(1-methylethyl)phenyl] methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine dihydrochloride; Biozol Diagnostica, Eching, Germany) was dissolved in waterfree 100% dimethyl sulfoxide (Acros Organics, Geel, Belgium; Ahn et al., ).

Techniques: Cell Culture, Staining, Functional Assay

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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Apolipoprotein E Receptor 2 Mediates Activated Protein C–Induced Endothelial Akt Activation and Endothelial Barrier Stabilization

doi: 10.1161/atvbaha.115.306795

Figure Lengend Snippet: Figure 1. Activated protein C (APC) requires Apolipoprotein E receptor 2 (ApoER2) in addition to endothelial protein C receptor (EPCR) and protease activated receptor (PAR)-1 for its antiapoptotic activity. A, Endothelial cells transfected with control small interfering RNA (siRNA) or siRNA specific for EPCR, PAR1, or ApoER2 were exposed to proapoptotic staurosporine followed by detected of apoptosis by the APO percentage assay. *P<0.0001 (2-tailed t test). B, Endothelial cells transfected with control siRNA or siRNA specific for EPCR, PAR1, or ApoER2 were exposed to tumor necrosis factor (TNF)-α, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay. *P<0.0002 (2-tailed t test). C, APC (20 nmol/L) was preincubated with soluble EPCR (sEPCR, 1 μmol/L), sE86A-EPCR (1 μmol/L), sApoER2 (1 μmol/L), or control buffer for 30 min before addition to cells; then staurosporine-induced apoptosis assays were done. *P<0.02. D, APC (20 nmol/L) was preincubated with sApoER2 (1 μmol/L), soluble very low-density lipo- protein receptor (sVLDLR; 1 μmol/L) or control buffer for 30 min before addition to cells and then TNFα-induced apoptosis assays were made. *P<0.001. E, Representative fluorescent microscopy images are shown for assay mixtures from TNFα-induced apoptosis assays as described in D.

Article Snippet: Materials - Sources for materials were as follows: ApoER2 siRNA (5’- UUGGUGAAGAUUAGGGAUG-3’) and control siRNA, Applied Bio System, Carlsbad, CA; EPCR siRNA (sc-39932) and PAR1 siRNA (sc-36663), Santa Cruz Biotech, USA; PAR1 antagonist SCH79797, Src kinase inhibitor PP2 and its analog PP3 and PI3K inhibitor LY294002 with its analogue Ly303511, Tocris Biosciences, Minneapolis, MN; thrombin and plasma-derived APC, (ERL) Enzyme Research Laboratories, South Blend, IN; mouse antiERK1/2 (3A7), rabbit anti-pThr202/Tyr204-ERK1/2 (197G2), mouse anti-Akt (40D4), rabbit antipSer473Akt (D9E), mouse anti-GSK3β (3D10), rabbit anti-pSer9-GSK3β (D3A4) and rabbit antipY232-Dab1, Cell Signaling Technology, San Diego, CA; rabbit monoclonal antibodies (MoAbs) anti-ApoER2 and anti-Dab1, Epitomics, Burlingame, CA; Infrared dye-conjugated secondary antibodies, LI-COR Biosciences, Lincoln, NE; and cellular media (Opti-MEM and D-MEM) and FITC dextran, Life Technologies, Carlsbad, CA.

Techniques: Activity Assay, Transfection, Control, Small Interfering RNA, TUNEL Assay, Microscopy