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MedChemExpress par1 inhibitor
Par1 Inhibitor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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par1 inhibitor - by Bioz Stars, 2026-05

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Figure 2. PAR1 response in cultured human renal mesangial cells (HRMCs). A, 697

Journal: Function (Oxford, England)

Article Title: PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

doi: 10.1093/function/zqae030

Figure Lengend Snippet: Figure 2. PAR1 response in cultured human renal mesangial cells (HRMCs). A, 697

Article Snippet: For PAR1 inhibition, we preincubated cells 146 with 10 μM PAR1 selective inhibitor RWJ 56110 (Tocris Bioscience, #2614) (water 147 D ow nloaded from https://academ ic.oup.com /function/advance-article/doi/10.1093/function/zqae030/7690769 by guest on 14 July 2024 O R IG IN A L U N E D IT E D M A N U S C R IP T soluble)27.

Techniques: Cell Culture

Figure 3. Protease-activated receptor 1 (PAR1) signaling promotes intracellular 710

Journal: Function (Oxford, England)

Article Title: PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

doi: 10.1093/function/zqae030

Figure Lengend Snippet: Figure 3. Protease-activated receptor 1 (PAR1) signaling promotes intracellular 710

Techniques:

Figure 5. The activation of protease-activated receptor 1 (PAR1) is required for 739

Journal: Function (Oxford, England)

Article Title: PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

doi: 10.1093/function/zqae030

Figure Lengend Snippet: Figure 5. The activation of protease-activated receptor 1 (PAR1) is required for 739

Techniques: Activation Assay

Figure 7. Pharmacological inhibition of PAR1-mediated oscillations in human 773

Journal: Function (Oxford, England)

Article Title: PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

doi: 10.1093/function/zqae030

Figure Lengend Snippet: Figure 7. Pharmacological inhibition of PAR1-mediated oscillations in human 773

Techniques: Inhibition

Figure 8. G-protein-coupled receptors PAR1 signaling activate TRPC3 and 791

Journal: Function (Oxford, England)

Article Title: PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

doi: 10.1093/function/zqae030

Figure Lengend Snippet: Figure 8. G-protein-coupled receptors PAR1 signaling activate TRPC3 and 791

Techniques:

Figure 9. Saturated PAR1 agonist concentrations, in addition to SR/ER calcium release, 814

Journal: Function (Oxford, England)

Article Title: PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

doi: 10.1093/function/zqae030

Figure Lengend Snippet: Figure 9. Saturated PAR1 agonist concentrations, in addition to SR/ER calcium release, 814

Techniques:

Effect of PAR1 blockage on hippocampal neuronal survival in glutamate toxicity and APC/AP9 treatment. Cells were cultured in NBM+ during days 9–10. Excitotoxicity was reached by substitution of NBM+ with buffered saline solutions (HBSS) containing glutamate (100 µM) for 40 min at 37 °C. PAR1 blocker SCH79797 (50 nM) was added to HBSS 30 min prior to Glu; APC (10 nM) or AP9 (20 µM) were added to HBSS 15 min prior to Glu. After 24 h, NBM+ was removed and cell viability was detected with the MTT assay kit as described in . Values are expressed as the mean ± S.D. of triplicate cultures. *— p < 0.05 compared to control group, #— p < 0.05 compared to group with glutamate (contr—with control HBSS containing 145 mM NaCl; 5 mM KCl; 1.8 mM CaCl 2 ; 1.0 mM MgCl 2 ; 20 mM HEPES; 5 mM glucose (pH 7.4), SCH—SCH79797).

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity

doi: 10.3390/ijms25021221

Figure Lengend Snippet: Effect of PAR1 blockage on hippocampal neuronal survival in glutamate toxicity and APC/AP9 treatment. Cells were cultured in NBM+ during days 9–10. Excitotoxicity was reached by substitution of NBM+ with buffered saline solutions (HBSS) containing glutamate (100 µM) for 40 min at 37 °C. PAR1 blocker SCH79797 (50 nM) was added to HBSS 30 min prior to Glu; APC (10 nM) or AP9 (20 µM) were added to HBSS 15 min prior to Glu. After 24 h, NBM+ was removed and cell viability was detected with the MTT assay kit as described in . Values are expressed as the mean ± S.D. of triplicate cultures. *— p < 0.05 compared to control group, #— p < 0.05 compared to group with glutamate (contr—with control HBSS containing 145 mM NaCl; 5 mM KCl; 1.8 mM CaCl 2 ; 1.0 mM MgCl 2 ; 20 mM HEPES; 5 mM glucose (pH 7.4), SCH—SCH79797).

Article Snippet: Human APC, NaCl, KCl, CaCl 2 , MgCl 2 , KH 2 PO 4 , HEPES, glucose, glutamate, NMDA, Ara C and PAR1 inhibitor SCH79797 were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, Saline, MTT Assay

APC and AP9 restore the Glu- and NMDA-induced [Ca 2+ ]i dysregulation in hippocampal neurons. ( A ) Glutamate-induced [Ca 2+ ]i alterations in cultured neurons and ( B ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of glutamate and PAR1 agonists), ( C ) NMDA-induced [Ca 2+ ]i alterations in cultured neurons and ( D ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of NMDA and PAR1 agonists). For [Ca 2+ ]i measurement, the cells were loaded with high-affinity Ca 2+ indicator Fluo-4 in the form of the acetoxymethyl (AM) ester (1–2 μM Fluo-4, 40 min, 37 °C). Fluo-4 fluorescence was excited at 488 nm and monitored at 505–535 nm. All measurements were carried out at 27–29 °C in HBSS. Glu or NMDA were washed out by a nominally calcium-free solution containing 0.1 mM EGTA instead of CaCl 2 and 2 mM MgCl 2 . For the measurement of the maximal signal at the final part of the experiments, a Ca 2+ ionophore ionomycin (2 μM) was applied in the presence of 5 mM Ca 2+ to saturate the indicator with Ca 2+ . *— p < 0.05 APC pretreatment compared to glutamate, **— p < 0.05 AP9 pretreatment compared to glutamate, #— p < 0.05 AP9 pretreatment compared to NMDA.

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity

doi: 10.3390/ijms25021221

Figure Lengend Snippet: APC and AP9 restore the Glu- and NMDA-induced [Ca 2+ ]i dysregulation in hippocampal neurons. ( A ) Glutamate-induced [Ca 2+ ]i alterations in cultured neurons and ( B ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of glutamate and PAR1 agonists), ( C ) NMDA-induced [Ca 2+ ]i alterations in cultured neurons and ( D ) changes of the areas under curves measured with Ca-dependent fluorescence Fluo-4 (effects of NMDA and PAR1 agonists). For [Ca 2+ ]i measurement, the cells were loaded with high-affinity Ca 2+ indicator Fluo-4 in the form of the acetoxymethyl (AM) ester (1–2 μM Fluo-4, 40 min, 37 °C). Fluo-4 fluorescence was excited at 488 nm and monitored at 505–535 nm. All measurements were carried out at 27–29 °C in HBSS. Glu or NMDA were washed out by a nominally calcium-free solution containing 0.1 mM EGTA instead of CaCl 2 and 2 mM MgCl 2 . For the measurement of the maximal signal at the final part of the experiments, a Ca 2+ ionophore ionomycin (2 μM) was applied in the presence of 5 mM Ca 2+ to saturate the indicator with Ca 2+ . *— p < 0.05 APC pretreatment compared to glutamate, **— p < 0.05 AP9 pretreatment compared to glutamate, #— p < 0.05 AP9 pretreatment compared to NMDA.

Article Snippet: Human APC, NaCl, KCl, CaCl 2 , MgCl 2 , KH 2 PO 4 , HEPES, glucose, glutamate, NMDA, Ara C and PAR1 inhibitor SCH79797 were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Cell Culture, Fluorescence

Biased signaling at PAR1 induced by APC and nanopeptide NPNDKYEPF-NH2 (AP9) analogs of the PAR1 tethered ligand liberated by APC. Though the exact mechanism underlying the neuroprotective effects of AP9 has not been discovered yet, it might function similarly to APC, promoting cytoprotective signaling via β-arrestin-2 and dishevelled-2 (Dvl-2) scaffold. In the present study, it was shown that the protective effects of APC and AP9 peptide are realized through stabilization of Glu- and NMDA-induced dysregulation of intracellular calcium concentration.

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity

doi: 10.3390/ijms25021221

Figure Lengend Snippet: Biased signaling at PAR1 induced by APC and nanopeptide NPNDKYEPF-NH2 (AP9) analogs of the PAR1 tethered ligand liberated by APC. Though the exact mechanism underlying the neuroprotective effects of AP9 has not been discovered yet, it might function similarly to APC, promoting cytoprotective signaling via β-arrestin-2 and dishevelled-2 (Dvl-2) scaffold. In the present study, it was shown that the protective effects of APC and AP9 peptide are realized through stabilization of Glu- and NMDA-induced dysregulation of intracellular calcium concentration.

Article Snippet: Human APC, NaCl, KCl, CaCl 2 , MgCl 2 , KH 2 PO 4 , HEPES, glucose, glutamate, NMDA, Ara C and PAR1 inhibitor SCH79797 were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Concentration Assay

Figure 1—PAR1 expression in human glomeruli. A: Expression of PAR1 in human podocytes. Representative microphotograph of decap- sulated human glomerulus freshly isolated from kidney cortex. Immunolabeling demonstrates colocalization of PAR1 (green) and nephrin (red; podocyte-speciﬁc marker) proteins. Cell nuclei are blue. Merged and separate confocal images for PAR1 and nephrin are shown (top: scale bar is 50 mm; bottom, scale bar is 25 mm). B: Immunostaining for PAR1 expression of control and diabetic samples (top, scale bar is 100 mm; bottom, scale bar is 25 mm). Black rectangles in the top images highlight the speciﬁed areas, which are zoomed-in in the bottom images, demonstrating the PAR1 expression in glomeruli. Summary of protein expression levels in the group without diabetes compared with the group with diabetes (11.2 ± 3.7 vs. 21.7 ± 6%, n $ 29 regions of interest, N = 3 samples, ***P < 0.001).

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 1—PAR1 expression in human glomeruli. A: Expression of PAR1 in human podocytes. Representative microphotograph of decap- sulated human glomerulus freshly isolated from kidney cortex. Immunolabeling demonstrates colocalization of PAR1 (green) and nephrin (red; podocyte-speciﬁc marker) proteins. Cell nuclei are blue. Merged and separate confocal images for PAR1 and nephrin are shown (top: scale bar is 50 mm; bottom, scale bar is 25 mm). B: Immunostaining for PAR1 expression of control and diabetic samples (top, scale bar is 100 mm; bottom, scale bar is 25 mm). Black rectangles in the top images highlight the speciﬁed areas, which are zoomed-in in the bottom images, demonstrating the PAR1 expression in glomeruli. Summary of protein expression levels in the group without diabetes compared with the group with diabetes (11.2 ± 3.7 vs. 21.7 ± 6%, n $ 29 regions of interest, N = 3 samples, ***P < 0.001).

Article Snippet: PAR1-activating peptides TFLLRNH2 or TRAP-6 (#1464 and #3497; Tocris Bioscience), PAR1 selective inhibitor RWJ 56100 (#2614; Tocris Bioscience), thrombin (#T6884; Sigma-Aldrich), urokinase (#ab92604; Abcam), and TRPC6 blocker SAR7334 (#5831; Tocris Bioscience) were applied to the podocytes of freshly isolated glomeruli in diabetic T2DN, GK, and control Wistar rats or cultured human podocytes to test changes in [Ca21]i influx.

Techniques: Expressing, Isolation, Immunolabeling, Marker, Immunostaining, Control

Figure 2—Hyperglycemia-induced increase in PAR1 response in cultured human podocytes. A: Representative microphotographs show in- tracellular calcium ([Ca21]i) changes (Fluo-8 AM) in response to PAR1 selective agonist TFLLR-NH2 (10 mmol/L) in control and high-glucose conditions (30 mmol/L for 12 h). Scale bar is 100 mm. B: High-glucose–induced signiﬁcant elevation in [Ca21]i response to PAR1 activation (red line) to compare with control (black line); a.u., arbitrary units. C: The statistical summary shows the maximum amplitude and total cal- cium (area under the curve [AUC] in individual podocytes (n $ 35 for each group, t test, ***P < 0.001). D: Changes in intracellular calcium af- ter PAR1 agonist application (TFLLR-NH2, 10 mmol/L) in the presence or absence of PAR1 receptor blocker RWJ 56100 (5 mmol/L). The statistical summary shows a signiﬁcant difference between the maximum amplitude of the control response and the response with the pres- ence of RWJ (n $ 25 cells, t test, ***P < 0.001).

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 2—Hyperglycemia-induced increase in PAR1 response in cultured human podocytes. A: Representative microphotographs show in- tracellular calcium ([Ca21]i) changes (Fluo-8 AM) in response to PAR1 selective agonist TFLLR-NH2 (10 mmol/L) in control and high-glucose conditions (30 mmol/L for 12 h). Scale bar is 100 mm. B: High-glucose–induced signiﬁcant elevation in [Ca21]i response to PAR1 activation (red line) to compare with control (black line); a.u., arbitrary units. C: The statistical summary shows the maximum amplitude and total cal- cium (area under the curve [AUC] in individual podocytes (n $ 35 for each group, t test, ***P < 0.001). D: Changes in intracellular calcium af- ter PAR1 agonist application (TFLLR-NH2, 10 mmol/L) in the presence or absence of PAR1 receptor blocker RWJ 56100 (5 mmol/L). The statistical summary shows a signiﬁcant difference between the maximum amplitude of the control response and the response with the pres- ence of RWJ (n $ 25 cells, t test, ***P < 0.001).

Techniques: Cell Culture, Control, Activation Assay

Figure 3—PAR1-mediated remodeling of podocyte structures. A: Representative light microscopy imaging of PAR1-mediated remodel- ing of tight junctions in cultured human podocytes after TFLLR-NH2 (20 mmol/L) application. Scale bar is 50 mm. B: Effect of PAR1 receptor agonist TFLLR-NH2 on podocyte structures assessed with SICM. An example of podocyte lamellipodia before and after application of TFLLR-NH2 (20 mmol/L). The arrows show the direction of lamellipo- dium changes over time. Scale bar is 5 mm. C: Summary graph dem- onstrating that activation of PAR1 signaling promotes retraction of podocyte lamellipodium. Data are shown as a cell area deviation nor- malized to the time of TFLLR-NH2 application (0 min). The graph dem- onstrates signiﬁcant retraction of the lamellipodium and a signiﬁcant decrease in cell surface area (n = 5, t test, P < 0.05). The arrows show the direction of lamellipodium changes over time.

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 3—PAR1-mediated remodeling of podocyte structures. A: Representative light microscopy imaging of PAR1-mediated remodel- ing of tight junctions in cultured human podocytes after TFLLR-NH2 (20 mmol/L) application. Scale bar is 50 mm. B: Effect of PAR1 receptor agonist TFLLR-NH2 on podocyte structures assessed with SICM. An example of podocyte lamellipodia before and after application of TFLLR-NH2 (20 mmol/L). The arrows show the direction of lamellipo- dium changes over time. Scale bar is 5 mm. C: Summary graph dem- onstrating that activation of PAR1 signaling promotes retraction of podocyte lamellipodium. Data are shown as a cell area deviation nor- malized to the time of TFLLR-NH2 application (0 min). The graph dem- onstrates signiﬁcant retraction of the lamellipodium and a signiﬁcant decrease in cell surface area (n = 5, t test, P < 0.05). The arrows show the direction of lamellipodium changes over time.

Techniques: Light Microscopy, Imaging, Cell Culture, Activation Assay

Figure 4—Downstream signaling changes in cultured human podo- cytes after PAR1 activation. Stimulation of PAR1 receptor cascade by the speciﬁc peptide TFLLR-NH2 promotes time-dependent remodeling in intracellular signaling pathways of the cultured human podocytes. A: Western blot analysis of PAR1, TRPC6, p-ERK1/2, and PLC-g1 in cultured human podocytes following activation of PAR1 signaling. B: The summary of time-dependent changes in PAR1 and TRPC6 ex- pression in podocytes stimulated by the 10 mmol/L TFLLR-NH2. Data were normalized to the baseline expression level of the corresponding pathway before PAR1 activation.

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 4—Downstream signaling changes in cultured human podo- cytes after PAR1 activation. Stimulation of PAR1 receptor cascade by the speciﬁc peptide TFLLR-NH2 promotes time-dependent remodeling in intracellular signaling pathways of the cultured human podocytes. A: Western blot analysis of PAR1, TRPC6, p-ERK1/2, and PLC-g1 in cultured human podocytes following activation of PAR1 signaling. B: The summary of time-dependent changes in PAR1 and TRPC6 ex- pression in podocytes stimulated by the 10 mmol/L TFLLR-NH2. Data were normalized to the baseline expression level of the corresponding pathway before PAR1 activation.

Techniques: Cell Culture, Activation Assay, Protein-Protein interactions, Western Blot, Expressing

$Figure 5—Acute serine protease application promotes activation of intracellular calcium in T2DN rat glomerular podocytes. A: The representative confocal imaging showed an application of thrombin (10 mmol/L) and a corresponding elevation of intracellular calcium (3 min between images) indi- cated as the ratio of Fluo-4 (green) and Fura 2TH (red) ﬂuorescent dyes. Scale bar is 20 mmol/L. B: Summary of the [Ca21]i amplitudes in response to thrombin in Wistar and T2DN rats (n $ 21, ***P < 0.001). C: Thrombin concentration in plasma of young Wistar (n = 8) and T2DN (n = 12) rats, and >48-week-old T2DN male (n = 12) and female (n = 8) rats (ANOVA, *P < 0.05). D: Summary of the [Ca21]i amplitudes in response to different concen- trations of urokinase in Wistar and T2DN rats (0.1 mg/mL, Wistar n = 10 vs. T2DN n = 20 cells, t test, ***P < 0.001; 2.7 mg/mL, Wistar n = 51 vs. T2DN n = 16 cells, t test, ***P < 0.001). E: Urokinase concentration in urine of 12-week-old Wistar and 12- and 48-week-old male and 48-week-old female T2DN rats (ANOVA, *P < 0.05). F: Western blot analysis of PAR1 expression in GK (n = 6), Wistar (n = 4), and young (n = 4) and old (n = 5) T2DN rats. Each band represents a fraction of freshly isolated glomeruli from one rat. Data are mean ± SEM, ANOVA, **P < 0.01, ***P < 0.001; a.u. arbitrary units.$

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 5—Acute serine protease application promotes activation of intracellular calcium in T2DN rat glomerular podocytes. A: The representative confocal imaging showed an application of thrombin (10 mmol/L) and a corresponding elevation of intracellular calcium (3 min between images) indi- cated as the ratio of Fluo-4 (green) and Fura 2TH (red) ﬂuorescent dyes. Scale bar is 20 mmol/L. B: Summary of the [Ca21]i amplitudes in response to thrombin in Wistar and T2DN rats (n $ 21, ***P < 0.001). C: Thrombin concentration in plasma of young Wistar (n = 8) and T2DN (n = 12) rats, and >48-week-old T2DN male (n = 12) and female (n = 8) rats (ANOVA, *P < 0.05). D: Summary of the [Ca21]i amplitudes in response to different concen- trations of urokinase in Wistar and T2DN rats (0.1 mg/mL, Wistar n = 10 vs. T2DN n = 20 cells, t test, ***P < 0.001; 2.7 mg/mL, Wistar n = 51 vs. T2DN n = 16 cells, t test, ***P < 0.001). E: Urokinase concentration in urine of 12-week-old Wistar and 12- and 48-week-old male and 48-week-old female T2DN rats (ANOVA, *P < 0.05). F: Western blot analysis of PAR1 expression in GK (n = 6), Wistar (n = 4), and young (n = 4) and old (n = 5) T2DN rats. Each band represents a fraction of freshly isolated glomeruli from one rat. Data are mean ± SEM, ANOVA, **P < 0.01, ***P < 0.001; a.u. arbitrary units.

Techniques: Activation Assay, Imaging, Concentration Assay, Clinical Proteomics, Western Blot, Expressing, Isolation

Figure 6—PAR-mediated TRPC6 activation. A: The progression of DN in T2DN rats (>48 weeks old) strongly correlates with increased TRPC6 expression (Wistar n = 3 vs. GK n = 3 vs. T2DN n = 6; mean ± SEM, ANOVA, **P < 0.01); a.u., arbitrary units. B: Changes in [Ca21]i in podocytes of freshly isolated glomeruli activated by acute application of selective PAR1 agonist TRAP-6 (10 mmol/L) after 10 min of preincubation with vehicle (DMSO; black) or TRPC6 blocker (20 mmol/L, SAR7334; red). The summary shows changes in maximum ﬂuorescence amplitude (Fluo-4 AM) in response to PAR1 activation rat freshly isolated glomeruli (n $ 7, t test, ***P < 0.001). C: The maximum amplitude ratio of [Ca21]i response in the podocyte of the freshly isolated glomerulus after PAR1 activation by TRAP-6 (10 mmol/L) was signiﬁ- cantly higher in T2DN rats compared with Wistar (n $ 22, t test, ***P < 0.001). In Wistar rats, both fast and slow calcium responses were ob- served (55% vs. 45%, respectively), but, in T2DN rats, only a slow, sustained response was present. D: Thrombin activates TRPC-like channels in the podocytes of the freshly isolated rat glomeruli from the Wistar rat. Microphotograph of the patch clamp experiment on the podocyte of the freshly isolated glomerulus (the white rectangle highlights the speciﬁed area shown on the photo below, showcasing the details of the patch-clamp procedure on the podocyte) and representative current trace from a cell. The application of thrombin (10 mmol/L) promoted a tran- sient increase in channel activity. The close state is indicated as “c,” and the open state is indicated as “o.”

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 6—PAR-mediated TRPC6 activation. A: The progression of DN in T2DN rats (>48 weeks old) strongly correlates with increased TRPC6 expression (Wistar n = 3 vs. GK n = 3 vs. T2DN n = 6; mean ± SEM, ANOVA, **P < 0.01); a.u., arbitrary units. B: Changes in [Ca21]i in podocytes of freshly isolated glomeruli activated by acute application of selective PAR1 agonist TRAP-6 (10 mmol/L) after 10 min of preincubation with vehicle (DMSO; black) or TRPC6 blocker (20 mmol/L, SAR7334; red). The summary shows changes in maximum ﬂuorescence amplitude (Fluo-4 AM) in response to PAR1 activation rat freshly isolated glomeruli (n $ 7, t test, ***P < 0.001). C: The maximum amplitude ratio of [Ca21]i response in the podocyte of the freshly isolated glomerulus after PAR1 activation by TRAP-6 (10 mmol/L) was signiﬁ- cantly higher in T2DN rats compared with Wistar (n $ 22, t test, ***P < 0.001). In Wistar rats, both fast and slow calcium responses were ob- served (55% vs. 45%, respectively), but, in T2DN rats, only a slow, sustained response was present. D: Thrombin activates TRPC-like channels in the podocytes of the freshly isolated rat glomeruli from the Wistar rat. Microphotograph of the patch clamp experiment on the podocyte of the freshly isolated glomerulus (the white rectangle highlights the speciﬁed area shown on the photo below, showcasing the details of the patch-clamp procedure on the podocyte) and representative current trace from a cell. The application of thrombin (10 mmol/L) promoted a tran- sient increase in channel activity. The close state is indicated as “c,” and the open state is indicated as “o.”

Techniques: Activation Assay, Expressing, Isolation, Patch Clamp, Activity Assay

Figure 8—The proposed PAR1-mediated signaling cascade in the diabetic kidney’s podocytes. Serine proteases, like thrombin, cause proteolytic cleavage of PAR1 and further activation of intracellular signaling in glomerular podocytes. The signaling mechanism, mediated by the canonical G protein coupling or engagement of b-arrestin–dependent pathways, promotes rapid transient calcium inﬂux through TRPC6 channels. Diabetic disease, characterized by marked elevation of serine proteases and hyperglycemia, causes constitutive activa- tion of PAR1, stimulates TRPC6 activity and expression, and leads to calcium overload and subsequent podocyte injury.

Journal: Diabetes

Article Title: Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

doi: 10.2337/db23-0032

Figure Lengend Snippet: Figure 8—The proposed PAR1-mediated signaling cascade in the diabetic kidney’s podocytes. Serine proteases, like thrombin, cause proteolytic cleavage of PAR1 and further activation of intracellular signaling in glomerular podocytes. The signaling mechanism, mediated by the canonical G protein coupling or engagement of b-arrestin–dependent pathways, promotes rapid transient calcium inﬂux through TRPC6 channels. Diabetic disease, characterized by marked elevation of serine proteases and hyperglycemia, causes constitutive activa- tion of PAR1, stimulates TRPC6 activity and expression, and leads to calcium overload and subsequent podocyte injury.

Techniques: Activation Assay, Activity Assay, Expressing

Determination of thrombin, PARs, and MIF expression at lesion sites following rat SCI. a ELISA measurement of thrombin protein levels at lesion sites following SCI at 0, 1, 4, and 7d. b Western blot analysis of MIF following SCI at 0, 1, 4, and 7d. Quantities were normalized to endogenous β-actin. c PCR assay for determining the expression abundance of par1 , par3, and par4 in the intact spinal cord. Quantities were normalized to endogenous gapdh . d–f RT-PCR assays of par1 ( d ), par3 ( e ), and par4 ( f ) transcriptional changes following SCI at 0, 1, 4, and 7d, respectively. Quantities were normalized to endogenous gapdh . Experiments were performed at least in technical triplicates. The values shown in the figures were the average of each technical replicate. Error bars represent the SEM (* P < 0.05). g Colocalization of PAR1 with GFAP-positive astrocytes before and after SCI. The sections were prepared using a cryostat from 0.25 cm length to the lesion epicenter. Rectangle indicates region magnified. Arrowheads indicate the positive signals. Scale bars, 500 μm, and 50 μm in magnification

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Determination of thrombin, PARs, and MIF expression at lesion sites following rat SCI. a ELISA measurement of thrombin protein levels at lesion sites following SCI at 0, 1, 4, and 7d. b Western blot analysis of MIF following SCI at 0, 1, 4, and 7d. Quantities were normalized to endogenous β-actin. c PCR assay for determining the expression abundance of par1 , par3, and par4 in the intact spinal cord. Quantities were normalized to endogenous gapdh . d–f RT-PCR assays of par1 ( d ), par3 ( e ), and par4 ( f ) transcriptional changes following SCI at 0, 1, 4, and 7d, respectively. Quantities were normalized to endogenous gapdh . Experiments were performed at least in technical triplicates. The values shown in the figures were the average of each technical replicate. Error bars represent the SEM (* P < 0.05). g Colocalization of PAR1 with GFAP-positive astrocytes before and after SCI. The sections were prepared using a cryostat from 0.25 cm length to the lesion epicenter. Rectangle indicates region magnified. Arrowheads indicate the positive signals. Scale bars, 500 μm, and 50 μm in magnification

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Functional annotation of DEGs following astrocyte stimulation with thrombin. a Primary cultured rat spinal cord astrocytes stained with GFAP and Hoechst 33,342 with purity over 95%. b PCR assay for determining the abundance of par1 , par3, and par4 at transcriptional levels in the primary astrocytes. Quantities were normalized to endogenous gapdh . c Bar graphs of DEGs following astrocyte stimulation with 100 nM thrombin for 6, 12, and 24 h, respectively. d Integration of DEGs at 6, 12, and 24 h. e GO analysis of the DEGs relating to biological processes. Scale bar, 50 μm in a

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Functional annotation of DEGs following astrocyte stimulation with thrombin. a Primary cultured rat spinal cord astrocytes stained with GFAP and Hoechst 33,342 with purity over 95%. b PCR assay for determining the abundance of par1 , par3, and par4 at transcriptional levels in the primary astrocytes. Quantities were normalized to endogenous gapdh . c Bar graphs of DEGs following astrocyte stimulation with 100 nM thrombin for 6, 12, and 24 h, respectively. d Integration of DEGs at 6, 12, and 24 h. e GO analysis of the DEGs relating to biological processes. Scale bar, 50 μm in a

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Functional Assay, Cell Culture, Staining

Effects of interfering PAR1, PAR3, or PAR4 expression on the thrombin-induced MIF production of astrocytes. a Western blot analysis of MIF protein levels in the astrocytes treated by 0–5 μM PAR1 inhibitor SCH79797 for 24 h in the presence of 100 nM thrombin. b Quantification data as shown in a . Quantities were normalized to endogenous β-actin. c MTT assay of SCH79797 effects on the cell viability of the astrocytes. d Interference efficiency of siRNA oligonucleotide for PAR3 was measured by RT-PCR, and siRNA1 was used for the knockdown experiments. e Western blot analysis of MIF in the astrocytes following PAR3 knockdown for 24 h, prior to stimulation with 100 nM thrombin for 24 h. Scrambles were used as control. f Quantification data as shown in e . Quantities were normalized to endogenous β-actin. (g) Western blot analysis of MIF protein levels in the astrocytes treated by 0–100 μM PAR4 inhibitor tcy-NH 2 for 24 h in the presence of 100 nM thrombin. h Quantification data as shown in g . Quantities were normalized to endogenous β-actin. i MTT assay of tcy-NH 2 effects on the cell viability of the astrocytes. Experiments were performed in triplicates. Error bars represent the SEM (* P < 0.05)

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Effects of interfering PAR1, PAR3, or PAR4 expression on the thrombin-induced MIF production of astrocytes. a Western blot analysis of MIF protein levels in the astrocytes treated by 0–5 μM PAR1 inhibitor SCH79797 for 24 h in the presence of 100 nM thrombin. b Quantification data as shown in a . Quantities were normalized to endogenous β-actin. c MTT assay of SCH79797 effects on the cell viability of the astrocytes. d Interference efficiency of siRNA oligonucleotide for PAR3 was measured by RT-PCR, and siRNA1 was used for the knockdown experiments. e Western blot analysis of MIF in the astrocytes following PAR3 knockdown for 24 h, prior to stimulation with 100 nM thrombin for 24 h. Scrambles were used as control. f Quantification data as shown in e . Quantities were normalized to endogenous β-actin. (g) Western blot analysis of MIF protein levels in the astrocytes treated by 0–100 μM PAR4 inhibitor tcy-NH 2 for 24 h in the presence of 100 nM thrombin. h Quantification data as shown in g . Quantities were normalized to endogenous β-actin. i MTT assay of tcy-NH 2 effects on the cell viability of the astrocytes. Experiments were performed in triplicates. Error bars represent the SEM (* P < 0.05)

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Expressing, Western Blot, MTT Assay, Reverse Transcription Polymerase Chain Reaction

Determination of the phosphorylated activation of the MAPKs/NFκB signals in the astrocytes following stimulation with thrombin. a Western blot analysis of phosphorylation of ERK, P38, JNK kinase, and p65NFκB protein after astrocyte stimulation with 0–200 nM thrombin for 24 h. b–e Quantification data as shown in a . Quantities were normalized to endogenous β-actin. f Western blot analysis of phosphorylation of ERK, P38, JNK kinase, and p65NFκB protein after astrocyte treatment with 0–5 μM SCH79797 for 24 h in the presence of 100 nM thrombin. g–j Quantification data as shown in f . Quantities were normalized to endogenous β-actin. Experiments were performed in triplicates. Error bars represent the SEM (* P < 0.05)

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Determination of the phosphorylated activation of the MAPKs/NFκB signals in the astrocytes following stimulation with thrombin. a Western blot analysis of phosphorylation of ERK, P38, JNK kinase, and p65NFκB protein after astrocyte stimulation with 0–200 nM thrombin for 24 h. b–e Quantification data as shown in a . Quantities were normalized to endogenous β-actin. f Western blot analysis of phosphorylation of ERK, P38, JNK kinase, and p65NFκB protein after astrocyte treatment with 0–5 μM SCH79797 for 24 h in the presence of 100 nM thrombin. g–j Quantification data as shown in f . Quantities were normalized to endogenous β-actin. Experiments were performed in triplicates. Error bars represent the SEM (* P < 0.05)

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Activation Assay, Western Blot

Effects of PAR1 inhibition on the production of MIF following rat SCI. a Western blot analysis of MIF from 1 cm cord segments following injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites of the contused cord at 0, 1, 4, and 7d, respectively. b Quantification data as shown in a . Quantities were normalized to endogenous β-actin. Experiments were performed in triplicates. Error bars represent the SEM, * P < 0.05, # P < 0.05, two-way ANOVA with Tukey’s test. c–h Immunostaining for MIF expressed in S100β-positive astrocytes at 4d following SCH79797 treatment on the injured cord. Arrowheads indicate the positive signals. Scale bars, 500 μm in c , e , and g ; 50 μm in d , f , and h

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Effects of PAR1 inhibition on the production of MIF following rat SCI. a Western blot analysis of MIF from 1 cm cord segments following injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites of the contused cord at 0, 1, 4, and 7d, respectively. b Quantification data as shown in a . Quantities were normalized to endogenous β-actin. Experiments were performed in triplicates. Error bars represent the SEM, * P < 0.05, # P < 0.05, two-way ANOVA with Tukey’s test. c–h Immunostaining for MIF expressed in S100β-positive astrocytes at 4d following SCH79797 treatment on the injured cord. Arrowheads indicate the positive signals. Scale bars, 500 μm in c , e , and g ; 50 μm in d , f , and h

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Inhibition, Western Blot, Injection, Immunostaining

Effects of PAR1 inhibition on the recovery of motor function following rat SCI. a HE staining of the injured spinal cord at 21 d after injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites. b Quantification data as shown in a from eight animals each 3 sections. Lesion area was analyzed within 2000 μm either side of the lesion center based on the HE-negative proportion. c BBB score of hindlimbs analyzed two-way repeated measures ANOVA followed by Sidak's post hoc test at 0d, 7d, 14d, and 21d following intrathecal injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites. Error bars represent the SEM (* P < 0.05). Scale bars 1000 μm

Journal: Journal of Neuroinflammation

Article Title: Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury

doi: 10.1186/s12974-022-02488-w

Figure Lengend Snippet: Effects of PAR1 inhibition on the recovery of motor function following rat SCI. a HE staining of the injured spinal cord at 21 d after injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites. b Quantification data as shown in a from eight animals each 3 sections. Lesion area was analyzed within 2000 μm either side of the lesion center based on the HE-negative proportion. c BBB score of hindlimbs analyzed two-way repeated measures ANOVA followed by Sidak's post hoc test at 0d, 7d, 14d, and 21d following intrathecal injection of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 or vehicle at lesion sites. Error bars represent the SEM (* P < 0.05). Scale bars 1000 μm

Article Snippet: For drug delivery, a total of 4.5 μl of 5 mM PAR1 inhibitor SCH79797 (R&D systems) was slowly injected intrathecally, prior to the incision suture.

Techniques: Inhibition, Staining, Injection

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