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: Expressing, Standard Deviation, MANN-WHITNEY, Multiplex Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)

doi: 10.1371/journal.pntd.0008596

Figure Lengend Snippet: Neutrophils were pre-sensitized with selective antagonists of ERK (U0126), PAR-1 (SCH79797) and PAR-2 (GB-83) for 15 min, separately. Pre-sensitized neutrophils were stimulated with 25 μg of ECV for 180 min and cells were stained with Hoechst stain. Neutrophils were photographed under a microscope (A) and, NETs were quantitated and represented as percent NETosis (B) . The data represented as mean ± SEM. *p < 0.05, when compared ECV versus ECV + antagonists. The whole cell lysates were analyzed for the phosphorylated ERK, expression of PAD4 and citH3 using Western blotting (C) . The p-ERK and PAD4 bands were quantitated using β-actin as a loading control. The citH3 bands were quantitated using H3 as a loading control. Data are representative of two independent experiments.

Article Snippet: SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA).

Techniques: Staining, Microscopy, Expressing, Western Blot

Journal: PLoS Neglected Tropical Diseases

Article Title: Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)

doi: 10.1371/journal.pntd.0008596

Figure Lengend Snippet: Mice footpads (n = 5) were pre-treated without or with PAR-1 antagonist (SCH79797) for 15 min and followed by the injection of ECV (½LD 50 ; 1.10 mg/kg). Mice footpads were photographed from day 1 to day 8 (A) and tissue injury was measured manually on a scale of 1 to 5 (B) . Red arrow indicates edema and black arrow indicates tissue necrosis. Data are representative of two independent experiments.

Article Snippet: SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA).

Techniques: Injection

Journal: CytoJournal

Article Title: A disintegrin-like and metalloproteinase 15 facilitates glioblastoma proliferation and metastasis through activation of the protease-activated receptor 1

doi: 10.25259/Cytojournal_92_2024

Figure Lengend Snippet: Inhibition of ADAM15-induced proliferation, migration, and invasion by PAR-1 antagonist treatment. (a) Evaluation by scratch assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. Scale bar = 100 μm. Objective: 100×. (b) Relative migration rate for scratch assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. (c) Evaluation by transwell assay with Matrigel invasion for 72 h. Scale bar = 50 μm. Objective: 200×. (d) Relative invasion rate for transwell assay after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. (e) Clonogenic assay in U251 and U87. (f) Measurement of colony number after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment. n = 5 independent replicates. ns: No significant, ✶ ✶ ✶ P < 0.001. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1.

Article Snippet: PAR1 antagonist: SCH79797 (Cat. No: SML1939, Merck Millipore, Billerica, MA, USA).

Techniques: Inhibition, Migration, Wound Healing Assay, Transwell Assay, Clonogenic Assay

Journal: CytoJournal

Article Title: A disintegrin-like and metalloproteinase 15 facilitates glioblastoma proliferation and metastasis through activation of the protease-activated receptor 1

doi: 10.25259/Cytojournal_92_2024

Figure Lengend Snippet: Inhibition of ADAM15-induced EMT by PAR-1 antagonist treatment. (a) Western blotting of E-cadherin, N-cadherin, and Vimentin protein expression after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment in U251. (b) Histogram of E-cadherin, N-cadherin, and Vimentin protein expression in U251. (c) Western blotting of E-cadherin, N-cadherin, and Vimentin protein expression after PAR-1 antagonist SCH79797 or PAR-2 antagonist FSLLRY-NH2 treatment in U87. (d) Histogram of E-cadherin, N-cadherin, and Vimentin protein expression in U87. (e) Immunofluorescence of E-cadherin and Vimentin staining in U251 and U87. Scale bar = 20 μm. Objective: 400×. n = 5 independent replicates. ns: Not significant, ✶ ✶ P < 0.01. ADAM15: A disintegrin-like and metalloproteinase 15, PAR1: Protease-activated receptor 1, Epithelial-mesenchymal transition.

Article Snippet: PAR1 antagonist: SCH79797 (Cat. No: SML1939, Merck Millipore, Billerica, MA, USA).

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Staining

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