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Tocris par1 antagonist sch 79797
Par1 Antagonist Sch 79797, supplied by Tocris, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par1+antagonist+sch+79797/sch79797/pm40550465-81-1-5
Average 90 stars, based on 1 article reviews

par1 antagonist sch 79797 - by Bioz Stars, 2026-09

90/100 stars

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Article Title: Proteinase-activated receptor-1 is an anti-inflammatory signal for colitis mediated by a type 2 immune response.
Article Snippet: Background: Activation of colonic proteinase activated receptor-1 (PAR1) provokes colonic inflammation and increases mucosal permeability in mice.. The mechanism of inflammation is not neurogenic like in the paw of rats but depends on PAR1-mediated activation monocytic cells.. PAR1 activation in the colon increases the release of lymphocyte T helper-1 (TH1) cytokines.

Article Title: Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration.
Article Snippet: The specific PAR1 antagonist SCH 79797 (10 M, Tocris, Avonmouth, UK) [21] and the I B kinase (IKK) inhibitor acety-11-keto- -boswellic acid (AK BA; 10 M) [20] were added 20 min prior to stimulation with thrombin (3 U/ml).

Inhibition:

Article Title: Protease activated receptor-1, PAR1, promotes placenta trophoblast invasion and beta-catenin stabilization.
Article Snippet: Despite extensive efforts toward elucidation of the molecular pathway controlling cytotrophoblast (CTB) invasion to the uterine decidua, it remains poorly defined.. There are striking similarities between tumor cell invasion and cytotrophoblast implantation to the deciduas whereby the role of Protease Activated Receptors (PARs) and wnt signaling is well recognized.. We examine here consequences of modulation of PAR1 and PAR2 expression and function on CTB invasion and b-catenin stabilization.

Concentration Assay:

Article Title: Intrinsic activation of the coagulation pathway induces mouse sciatic nerve hypoexcitability.
Article Snippet: Thrombin (T4648, Sigma-Aldrich, St. Louis, MO, USA) was applied at a concentration of 200 U/ml. .. A PAR1 antagonist (SCH 79797, Tocris Bioscience, Bristol, United Kingdom) was applied at a concentration of 1 μM. ..

MTT Assay:

Article Title: Effect of enteropeptidase on survival of cultured hippocampal neurons under conditions of glutamate toxicity.
Article Snippet: The effects of full size bovine enteropeptidase (BEK) and of human recombinant light chain enteropeptidase (L HEP) on survival of cultured hippocampal neurons were studied under conditions of glutamate excitotoxicity.. Low con centrations of L HEP or BEK (0.1 1 and 0.1 0.5 nM, respectively) protected hippocampal neurons against the death caused by 100 μM glutamate.. Using the PAR1 (proteinase activated receptor) antagonist SCH 79797, we revealed a PAR1 dependent mechanism of neuroprotective action of low concentrations of enteropeptidase.

Image Search Results

FIGURE 1 | Lower LTP in pilocarpine injected animals is prevented by speciﬁc thrombin inhibition. (A) LTP was signiﬁcantly reduced in pilocarpine treated animals compared to control. Both the speciﬁc thrombin inhibitor NAPAP (B) and the PAR1 inhibitor SCH79797 (C) prevented LTP reduction (n = 8 for each group).

Journal: Frontiers in cellular neuroscience

Article Title: Modulation of the Thrombin Pathway Restores LTP in a Pilocarpine Mice Model of Status Epilepticus.

doi: 10.3389/fncel.2022.900925

Figure Lengend Snippet: FIGURE 1 | Lower LTP in pilocarpine injected animals is prevented by speciﬁc thrombin inhibition. (A) LTP was signiﬁcantly reduced in pilocarpine treated animals compared to control. Both the speciﬁc thrombin inhibitor NAPAP (B) and the PAR1 inhibitor SCH79797 (C) prevented LTP reduction (n = 8 for each group).

Article Snippet: To test the effects of thrombin or PAR1 inhibition, animals were i.p. injected with a single dose of the thrombin inhibitor α-NAPAP [Nα-(2-naphthalenesulfonyl glycyl)-4-amidino-DL-phenylalaninepiperidide, Santa Cruz, 0.75 mg/kg in phosphate-buffered saline (PBS), SC208083] or the PAR1 antagonist SCH79797 (Tocris Bioscience, 1592, 25 μg/kg in PBS, prepared immediately before use) 15 min before the atropine injection.

Techniques: Injection, Inhibition, Control

FIGURE 2 | Coagulation and inﬂammation mRNA expression modiﬁcation in the hippocampus of animals 24 h after SE induction. (A) PAR1 levels increased signiﬁcantly in the pilocarpine-only group compared to the SCH79797 PAR1 antagonist group. No signiﬁcant differences between groups were measured in prothrombin (B) relative expression. Levels of the inﬂammatory marker TNF-α (C) were elevated following SE induction in the pilocarpine-only group compared to controls (Coagulation and inﬂammatory markers: control n = 8, pilocarpine n = 7, α-NAPAP n = 8, SCH79797 n = 7). SE, status epilepticus; PAR1, protease-activated receptor; TNF-α, tumor necrosis factor α. *p < 0.05, **p < 0.01.

Journal: Frontiers in cellular neuroscience

Article Title: Modulation of the Thrombin Pathway Restores LTP in a Pilocarpine Mice Model of Status Epilepticus.

doi: 10.3389/fncel.2022.900925

Figure Lengend Snippet: FIGURE 2 | Coagulation and inﬂammation mRNA expression modiﬁcation in the hippocampus of animals 24 h after SE induction. (A) PAR1 levels increased signiﬁcantly in the pilocarpine-only group compared to the SCH79797 PAR1 antagonist group. No signiﬁcant differences between groups were measured in prothrombin (B) relative expression. Levels of the inﬂammatory marker TNF-α (C) were elevated following SE induction in the pilocarpine-only group compared to controls (Coagulation and inﬂammatory markers: control n = 8, pilocarpine n = 7, α-NAPAP n = 8, SCH79797 n = 7). SE, status epilepticus; PAR1, protease-activated receptor; TNF-α, tumor necrosis factor α. *p < 0.05, **p < 0.01.

Techniques: Coagulation, Expressing, Marker, Control

FIGURE 3 | Elevation in serum marker for neuronal damage, prevented by thrombin PAR1 pathway inhibition. (A) Serum levels of NfL, a marker for neuronal damage, were elevated in pilocarpine only animals. Thrombin PAR1 pathway inhibitors prevented the increased NfL levels. (B) Systemic inﬂammation as evaluated by TNF-α levels was similar in all four groups. *p < 0.05.

Journal: Frontiers in cellular neuroscience

Article Title: Modulation of the Thrombin Pathway Restores LTP in a Pilocarpine Mice Model of Status Epilepticus.

doi: 10.3389/fncel.2022.900925

Figure Lengend Snippet: FIGURE 3 | Elevation in serum marker for neuronal damage, prevented by thrombin PAR1 pathway inhibition. (A) Serum levels of NfL, a marker for neuronal damage, were elevated in pilocarpine only animals. Thrombin PAR1 pathway inhibitors prevented the increased NfL levels. (B) Systemic inﬂammation as evaluated by TNF-α levels was similar in all four groups. *p < 0.05.

Techniques: Marker, Inhibition

Thrombin activity and protease activated receptor 1 (PAR1) levels in superoxide dismutase 1 (SOD1 mice): ( A ) thrombin activity is significantly increased in the posterior brain slices, as well as posterior frontal cortex (slice 11, 6, p < 0.001, p < 0.05, respectively). Measured from the anterior slice number 3 (#3) to the posterior slice number 11 (#11). Number of animals: SOD1 n = 11, healthy control n = 7; ( B ) summation of thrombin activity in all brain slices of SOD1 mice is significantly elevated compared to healthy control mice ( p = 0.013). Number of animals: SOD1 n = 11, healthy control n = 7; ( C ) PAR1 levels, as measured by western blot, are significantly lower in SOD1 mice brains compared to healthy control mice ( p < 0.01). Number of animals: SOD1 n = 4, healthy control n = 5; ( D ) PAR1 levels in spinal cords of SOD1 mice were lower compared to healthy control mice. Number of animals: SOD1 n = 5, healthy control n = 4. * p < 0.05, ** p < 0.01. Illustration created using BioRender.com.

Journal: International Journal of Molecular Sciences

Article Title: Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms21103419

Figure Lengend Snippet: Thrombin activity and protease activated receptor 1 (PAR1) levels in superoxide dismutase 1 (SOD1 mice): ( A ) thrombin activity is significantly increased in the posterior brain slices, as well as posterior frontal cortex (slice 11, 6, p < 0.001, p < 0.05, respectively). Measured from the anterior slice number 3 (#3) to the posterior slice number 11 (#11). Number of animals: SOD1 n = 11, healthy control n = 7; ( B ) summation of thrombin activity in all brain slices of SOD1 mice is significantly elevated compared to healthy control mice ( p = 0.013). Number of animals: SOD1 n = 11, healthy control n = 7; ( C ) PAR1 levels, as measured by western blot, are significantly lower in SOD1 mice brains compared to healthy control mice ( p < 0.01). Number of animals: SOD1 n = 4, healthy control n = 5; ( D ) PAR1 levels in spinal cords of SOD1 mice were lower compared to healthy control mice. Number of animals: SOD1 n = 5, healthy control n = 4. * p < 0.05, ** p < 0.01. Illustration created using BioRender.com.

Article Snippet: TLCK ( N -Tosyl-Lys-chloromethylketone) was used at a dose of 4.4 mg/kg and PAR1 antagonist (SCH-79797, Tocris, Bristol, UK) was used at a dose of 25 μg/kg, both were administered by intraperitoneal injections ( ).

Techniques: Activity Assay, Control, Western Blot

PAR1 localization in mice cerebellum and cortex: Confocal fluorescent microscopy immunohistochemistry with antibodies labeling PAR1 (magenta) and the astrocytic marker glial acidic fibrillary protein (GFAP, green) in ( A – H ) the cerebellum and ( I – P ) the cortex of healthy and SOD1 mice in ×20 magnification. Co-localization of PAR1 with GFAP is seen in pink. Further magnification of ×63 is shown in C,G,K,O. High power magnification of selected areas (indicated by white frames) are shown in D,H,L,P. SOD1 mice show reduced levels of PAR1, and disruption of its co-localization with GFAP, as was indicated by qualitative analysis. Reduction of GFAP staining suggests astrocytic morphological modification. Immunohistochemistry was performed at the age of 20 weeks. All photos had color adaptation using Adobe Photoshop CS5. Number of animals: SOD1 n = 3, healthy control n = 4.

Journal: International Journal of Molecular Sciences

Article Title: Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms21103419

Figure Lengend Snippet: PAR1 localization in mice cerebellum and cortex: Confocal fluorescent microscopy immunohistochemistry with antibodies labeling PAR1 (magenta) and the astrocytic marker glial acidic fibrillary protein (GFAP, green) in ( A – H ) the cerebellum and ( I – P ) the cortex of healthy and SOD1 mice in ×20 magnification. Co-localization of PAR1 with GFAP is seen in pink. Further magnification of ×63 is shown in C,G,K,O. High power magnification of selected areas (indicated by white frames) are shown in D,H,L,P. SOD1 mice show reduced levels of PAR1, and disruption of its co-localization with GFAP, as was indicated by qualitative analysis. Reduction of GFAP staining suggests astrocytic morphological modification. Immunohistochemistry was performed at the age of 20 weeks. All photos had color adaptation using Adobe Photoshop CS5. Number of animals: SOD1 n = 3, healthy control n = 4.

Techniques: Microscopy, Immunohistochemistry, Labeling, Marker, Disruption, Staining, Modification, Control

Body weight, rotarod and survival outcomes of SOD1 mice with various treatment: ( A ) healthy control mice progressively gain weight, while SOD1 mice gain weight up until the age of 11 weeks. Following a plateau, at the age of 17 weeks, SOD1 mice show progressive weight loss. A significant difference in body weight is measured between healthy control and SOD1 mice. This difference persists throughout the remaining experiment duration ( p < 0.0001). In the insert, an enlargement of weeks 16–20 is presented. All treatments show a beneficial effect on weight loss. Number of animals: healthy control n = 6, SOD1 n = 22, N -Tosyl-Lys-chloromethylketone (TLCK) n = 21, PAR1 antagonist n = 14, S-trans-trans-farnesylthiosalicylic acid (FTS-40) n = 21, FTS-25 n = 14; ( B ) SOD1 mice show shorter rotarod scores starting at the age of 14 weeks and subsequently thorough the experiment ( p < 0.0001). FTS-40 improves rotarod scores at weeks 15 and 16 ( p < 0.01). Number of animals: Healthy control n = 5, SOD1 n = 21, TLCK n = 20, PAR1 antagonist n = 14, FTS-40 n = 5, FTS-25 n = 13; ( C ) Survival is significantly improved by all treatments except for high-dose FTS ( p < 0.01). Number of animals: Healthy control n = 6, SOD1 n = 22, PAR1 antagonist n = 13, TLCK n = 19, FTS-25 n = 14, FTS-40 n = 21, FTS-60 n = 19. * p < 0.05.

Journal: International Journal of Molecular Sciences

Article Title: Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms21103419

Figure Lengend Snippet: Body weight, rotarod and survival outcomes of SOD1 mice with various treatment: ( A ) healthy control mice progressively gain weight, while SOD1 mice gain weight up until the age of 11 weeks. Following a plateau, at the age of 17 weeks, SOD1 mice show progressive weight loss. A significant difference in body weight is measured between healthy control and SOD1 mice. This difference persists throughout the remaining experiment duration ( p < 0.0001). In the insert, an enlargement of weeks 16–20 is presented. All treatments show a beneficial effect on weight loss. Number of animals: healthy control n = 6, SOD1 n = 22, N -Tosyl-Lys-chloromethylketone (TLCK) n = 21, PAR1 antagonist n = 14, S-trans-trans-farnesylthiosalicylic acid (FTS-40) n = 21, FTS-25 n = 14; ( B ) SOD1 mice show shorter rotarod scores starting at the age of 14 weeks and subsequently thorough the experiment ( p < 0.0001). FTS-40 improves rotarod scores at weeks 15 and 16 ( p < 0.01). Number of animals: Healthy control n = 5, SOD1 n = 21, TLCK n = 20, PAR1 antagonist n = 14, FTS-40 n = 5, FTS-25 n = 13; ( C ) Survival is significantly improved by all treatments except for high-dose FTS ( p < 0.01). Number of animals: Healthy control n = 6, SOD1 n = 22, PAR1 antagonist n = 13, TLCK n = 19, FTS-25 n = 14, FTS-40 n = 21, FTS-60 n = 19. * p < 0.05.

Techniques: Control

Timeline: transgenic mice were genotyped at birth. Weighing and rotarod evaluation were performed every week. Treatments were given at weeks 4–20, five times a week. Thrombin activity, western blot for PAR1 and immunostaining were conducted at the age of 20 weeks. In the survival arm of the study, mice were followed up until the age of 167 days.

Journal: International Journal of Molecular Sciences

Article Title: Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms21103419

Figure Lengend Snippet: Timeline: transgenic mice were genotyped at birth. Weighing and rotarod evaluation were performed every week. Treatments were given at weeks 4–20, five times a week. Thrombin activity, western blot for PAR1 and immunostaining were conducted at the age of 20 weeks. In the survival arm of the study, mice were followed up until the age of 167 days.

Techniques: Transgenic Assay, Activity Assay, Western Blot, Immunostaining

(A-C) Immunofluorescence of PAR1 (green) in myometrium from a pregnant woman (A) , non-pregnant woman ( B ), and fetal membrane (C) . Nuclei were stained with DAPI (blue). Am, amnion, Cho, chorion, Deci, decidua. (D-G) Localization of hemorrhage, thrombin, and PAR1 in placental abruption at 25 weeks of gestation (D and E) , and 33 weeks gestation (F and G) resulting in disseminated intravascular coagulopathy and uterine bleeding requiring hysterectomy for hemostasis. (D and F) Hematoxylin and eosin staining of the myometrium adjacent to the placenta. Note that hemorrhage infiltrated the myometrium. Bars, 50 μm. (E and G) Immunofluorescence of PAR1 (green), thrombin (red), and DAPI (blue) at the same location of (A) . Bars, 50 μm.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A-C) Immunofluorescence of PAR1 (green) in myometrium from a pregnant woman (A) , non-pregnant woman ( B ), and fetal membrane (C) . Nuclei were stained with DAPI (blue). Am, amnion, Cho, chorion, Deci, decidua. (D-G) Localization of hemorrhage, thrombin, and PAR1 in placental abruption at 25 weeks of gestation (D and E) , and 33 weeks gestation (F and G) resulting in disseminated intravascular coagulopathy and uterine bleeding requiring hysterectomy for hemostasis. (D and F) Hematoxylin and eosin staining of the myometrium adjacent to the placenta. Note that hemorrhage infiltrated the myometrium. Bars, 50 μm. (E and G) Immunofluorescence of PAR1 (green), thrombin (red), and DAPI (blue) at the same location of (A) . Bars, 50 μm.

Article Snippet: On the day of experiments, cells in collagen gels were pretreated with the following reagents for 1 h: 100 nM PAR1 selective antagonist SCH79797 (No.1592 Tocris), 1 μM ROCK inhibitor Y-27632 (10005583, Cayman Chemical), 10 μM MLCK inhibitor, ML-7 (11801, Cayman Chemical), 10 μM of indomethacin (I-7378, Sigma), or 1 μM of progesterone (28921–64, Nacalai-tesque).

Techniques: Immunofluorescence, Membrane, Staining

Thrombin increased contraction of primary human myometrial cells through PAR1. (A and B) (Left images) Representative images of collagen lattice assay of human myometrial cells at 30 min treated with PBS (Ctl) and thrombin (A) or PAR1 activating peptide, TFLLR (B) . (Right graphs) Quantification of myometrial contractions in collagen lattice assays (n = 3). **, p < 0.01 at each time point. (C) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with or without 100 nM PAR1 selective inhibitor (SCH79797, PAR1-i) for 1 h (n = 3). Representative image (upper panel) and quantification of gel areas (lower graph). The experiments were repeated three times. *, p < 0.05, and **, p < 0.01.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: Thrombin increased contraction of primary human myometrial cells through PAR1. (A and B) (Left images) Representative images of collagen lattice assay of human myometrial cells at 30 min treated with PBS (Ctl) and thrombin (A) or PAR1 activating peptide, TFLLR (B) . (Right graphs) Quantification of myometrial contractions in collagen lattice assays (n = 3). **, p < 0.01 at each time point. (C) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with or without 100 nM PAR1 selective inhibitor (SCH79797, PAR1-i) for 1 h (n = 3). Representative image (upper panel) and quantification of gel areas (lower graph). The experiments were repeated three times. *, p < 0.05, and **, p < 0.01.

Techniques:

(A) Immunocytochemistry of PAR1 (red) and phosphorylated MLC2 (Ser19, green) in human myometrial cells. (B, C) Immunoblots of phosphorylated MLC2 (p-MLC2), total MLC2, and β-actin of myometrial cells. Myometrial cells were treated with thrombin (2 U/mL) as a function of time (B) , or pretreated with 100 nM PAR1 inhibitor (SCH79797) for 1 h, and then treated with 2 U/mL of thrombin for 30 min (C) . The experiments were repeated three times.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A) Immunocytochemistry of PAR1 (red) and phosphorylated MLC2 (Ser19, green) in human myometrial cells. (B, C) Immunoblots of phosphorylated MLC2 (p-MLC2), total MLC2, and β-actin of myometrial cells. Myometrial cells were treated with thrombin (2 U/mL) as a function of time (B) , or pretreated with 100 nM PAR1 inhibitor (SCH79797) for 1 h, and then treated with 2 U/mL of thrombin for 30 min (C) . The experiments were repeated three times.

Techniques: Immunocytochemistry, Western Blot

(A) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with 1 μM progesterone (P4) for 1 h. Representative image (upper panel) and quantification of gel areas (lower graph). (B) Inhibition of thrombin-induced increases of PTGS2 , IL1B , and F2R mRNA by P4. Myometrial cells were pretreated with 1 μM of P4 for 1 h, and then treated with 2 U/mL of thrombin. (C) Gene expressions of progesterone receptor-A and–B ( PgR-A and PgR-B ) with 24 h treatment of 1 U/mL of thrombin, 10 nM of PGE2, and 10 nM of PGF2α (upper graphs) and PgR-A to PgR-B ratio (lower graphs). n = 3 in each group. *, p < 0.05, and **, p < 0.01. The experiments were repeated three times.

Journal: PLoS ONE

Article Title: Mechanisms of thrombin-Induced myometrial contractions: Potential targets of progesterone

doi: 10.1371/journal.pone.0231944

Figure Lengend Snippet: (A) Collagen lattice assay of myometrial cells at 30 min with 2 U/mL of thrombin (Thr) pretreated with 1 μM progesterone (P4) for 1 h. Representative image (upper panel) and quantification of gel areas (lower graph). (B) Inhibition of thrombin-induced increases of PTGS2 , IL1B , and F2R mRNA by P4. Myometrial cells were pretreated with 1 μM of P4 for 1 h, and then treated with 2 U/mL of thrombin. (C) Gene expressions of progesterone receptor-A and–B ( PgR-A and PgR-B ) with 24 h treatment of 1 U/mL of thrombin, 10 nM of PGE2, and 10 nM of PGF2α (upper graphs) and PgR-A to PgR-B ratio (lower graphs). n = 3 in each group. *, p < 0.05, and **, p < 0.01. The experiments were repeated three times.

Techniques: Inhibition

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

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.

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.

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

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.

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.

Techniques: Expressing, Gene Expression, Derivative Assay, Comparison

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.

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.

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

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

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.

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

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.

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.

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

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.

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.

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

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