Journal: iScience

Article Title: The role of protease-activated receptor 1 signaling in CD8 T cell effector functions

doi: 10.1016/j.isci.2021.103387

Figure Lengend Snippet: PAR1 signaling cross-talks with TCR-induced calcium mobilization in activated human CD8 T cells Human CD8 T cells isolated from healthy volunteers were activated with anti-CD3 and anti-CD28 mAbs and expanded in the presence of IL-2 for 11–18 days. Calcium mobilization was measured using FLIPR assay for up to 200 s. (A) Kinetics of TCR-induced calcium flux in response to 0.5 μg/mL and 1 μg/mL CD3 mAbs in the presence or absence of 3 nM thrombin. Representative experiment of (n = 3). (B) (Left panel) Representative experiment of TCR-induced calcium mobilization in activated human CD8 T cells in response to 1 μg/mL anti-CD3 mAb in the presence or absence of PAR1 inhibitors SCH79797 (2.5 μM) and SCH530348 (5 μM), PLC inhibitor 1 μM U73122, or 1 μM analog control U73343. The graph (right panel) represents the experiments of TCR-induced calcium mobilization with activated CD8 T cells from healthy donors tested (n = 12). Box and whisker plot represent median value with interquartile range. The total area under the curve (AUC) was calculated using GraphPad prism. Comparisons between culture conditions was performed using nonparametric paired Wilcoxon test. p value <0.05 was considered significant. (C) PAR1 surface expression upon TCR stimulation. Activated CD8 T cells from healthy donors (n = 8) were stimulated in media and plate bound anti-CD3 mAb at concentration 10 μg/mL for 30, 120, and 240 min, and PAR1 and CD107a surface expression was evaluated by flow cytometry. Representative contour plots of surface expression of PAR1 and CD107a. (D) Loss PAR1 surface staining over time of stimulation with 10 μg/mL anti-CD3 mAb during 30, 120, and 240 min (left panel) and stimulation with 1 μg/mL and 10 μg/mL anti-CD3 mAb for 240 min (right panel). The graph represents PAR1 surface staining as the frequency of Mean Fluorescence Intensity (MFI) of the stimulated condition relative to the expression PAR1 (MFI) in the unstimulated condition. (E) Surface expression of CD107a (MFI) during stimulation with 10 μg/mL anti-CD3 mAb. (F) Granzyme A was measured in the supernatants of the stimulated cells. Comparisons between culture conditions was performed using nonparametric paired Wilcoxon test. p value <0.05 was considered significant.

Article Snippet: After 30 min of incubation at 37°C and 5% CO 2 , media or the PAR1 inhibitors SCH530348 (Bio-techne/Tocris, MN) and SCH79797 (Axon Medchem, VA), PLC inhibitor U73122 and its analog U73343 (Sigma, MO) were added to the cells and they were further incubated another 20 min.

Techniques: Isolation, Control, Whisker Assay, Expressing, Concentration Assay, Flow Cytometry, Staining, Fluorescence

Journal: iScience

Article Title: The role of protease-activated receptor 1 signaling in CD8 T cell effector functions

doi: 10.1016/j.isci.2021.103387

Figure Lengend Snippet: PAR1 blockade in human CD8 T cells inhibits degranulation and cytotoxicity function by reducing MTOC positioning at the IS Human CD8 T cells from healthy donors (n = 9) were activated with CD3/CD28 for 3 days and followed by expansion for an additional 11–18 days in the presence of rhIL-2. Redirected killing assay was performed using biotinylated L1210 (FAS − ) targets. Target cells were labeled with CTFR. Both effectors and targets were labeled with NFL1 to exclude the dead cells in the flow cytometry analysis. Activated CD8 T cells were preincubated with media or 2.5 μM and 5 μM of the PAR1 inhibitor SCH79797 before mixing with the target cells at 27:1 (E:T ratio) in the presence or absence of 10 μg/mL anti-CD3 and GZB substrate. After 1-h incubation cells were harvested and stained for CD8 and CD107a. (A) Gating strategy to analyze target cells (CTFR + ) and effector CD8 T cells. (B) Targets were analyzed for GZB activity (CTFR + Substrate + ). (C) Effector CD8 T cells were analyzed for surface expression of lysosomal-associated membrane protein (LAMP-1, CD107a). (D) Quatitative plot of the effect of PAR1 inhibition by SCH79797 on the granzyme activity (CTFR + Substrate + ) in the targets. (E) Quantitative plot of the effect of PAR1 inhibition by SCH79797 on CD8 T cell degranulation as indicated by surface CD107a expression. Data are represented with box and whisker plot of median value with interquartile range. Statistical analysis was performed using nonparametric Mann-Whitney test for comparison between groups and nonparametric paired Wilcoxon test for comparisons between conditions. p value <0.05 was considered significant. (F) Conjugates were prepared using biotinylated L1210 and activated CD8 T cells in the presence of 10 μg/mL biotin-CD3. F-actin accumulation at the immunological synapse (IS) was measured by the average and integrated intensity of phalloidin staining. The box and whisker plot showed median value with interquartile range (n = 257–263 conjugates of each group). (G) Conjugates were stained with antibodies against alpha-tubulin (red) to visualize the microtubule-organizing center (MTOC). Quantification of MTOC polarization was expressed as distal (Di), proximal (P), and docked (Do) to the IS. Data are pooled from three independent experiments (n = 185–190 conjugates of each group).

Article Snippet: After 30 min of incubation at 37°C and 5% CO 2 , media or the PAR1 inhibitors SCH530348 (Bio-techne/Tocris, MN) and SCH79797 (Axon Medchem, VA), PLC inhibitor U73122 and its analog U73343 (Sigma, MO) were added to the cells and they were further incubated another 20 min.

Techniques: Labeling, Flow Cytometry, Incubation, Staining, Activity Assay, Expressing, Membrane, Inhibition, Whisker Assay, MANN-WHITNEY, Comparison

Journal: iScience

Article Title: The role of protease-activated receptor 1 signaling in CD8 T cell effector functions

doi: 10.1016/j.isci.2021.103387

Figure Lengend Snippet:

Article Snippet: After 30 min of incubation at 37°C and 5% CO 2 , media or the PAR1 inhibitors SCH530348 (Bio-techne/Tocris, MN) and SCH79797 (Axon Medchem, VA), PLC inhibitor U73122 and its analog U73343 (Sigma, MO) were added to the cells and they were further incubated another 20 min.

Techniques: Recombinant, Selection, Software

Journal: iScience

Article Title: The role of protease-activated receptor 1 signaling in CD8 T cell effector functions

doi: 10.1016/j.isci.2021.103387

Figure Lengend Snippet: PAR1 signaling cross-talks with TCR-induced calcium mobilization in activated human CD8 T cells Human CD8 T cells isolated from healthy volunteers were activated with anti-CD3 and anti-CD28 mAbs and expanded in the presence of IL-2 for 11–18 days. Calcium mobilization was measured using FLIPR assay for up to 200 s. (A) Kinetics of TCR-induced calcium flux in response to 0.5 μg/mL and 1 μg/mL CD3 mAbs in the presence or absence of 3 nM thrombin. Representative experiment of (n = 3). (B) (Left panel) Representative experiment of TCR-induced calcium mobilization in activated human CD8 T cells in response to 1 μg/mL anti-CD3 mAb in the presence or absence of PAR1 inhibitors SCH79797 (2.5 μM) and SCH530348 (5 μM), PLC inhibitor 1 μM U73122, or 1 μM analog control U73343. The graph (right panel) represents the experiments of TCR-induced calcium mobilization with activated CD8 T cells from healthy donors tested (n = 12). Box and whisker plot represent median value with interquartile range. The total area under the curve (AUC) was calculated using GraphPad prism. Comparisons between culture conditions was performed using nonparametric paired Wilcoxon test. p value <0.05 was considered significant. (C) PAR1 surface expression upon TCR stimulation. Activated CD8 T cells from healthy donors (n = 8) were stimulated in media and plate bound anti-CD3 mAb at concentration 10 μg/mL for 30, 120, and 240 min, and PAR1 and CD107a surface expression was evaluated by flow cytometry. Representative contour plots of surface expression of PAR1 and CD107a. (D) Loss PAR1 surface staining over time of stimulation with 10 μg/mL anti-CD3 mAb during 30, 120, and 240 min (left panel) and stimulation with 1 μg/mL and 10 μg/mL anti-CD3 mAb for 240 min (right panel). The graph represents PAR1 surface staining as the frequency of Mean Fluorescence Intensity (MFI) of the stimulated condition relative to the expression PAR1 (MFI) in the unstimulated condition. (E) Surface expression of CD107a (MFI) during stimulation with 10 μg/mL anti-CD3 mAb. (F) Granzyme A was measured in the supernatants of the stimulated cells. Comparisons between culture conditions was performed using nonparametric paired Wilcoxon test. p value <0.05 was considered significant.

Article Snippet: After 30 min of incubation at 37°C and 5% CO 2 , media or the PAR1 inhibitors SCH530348 (Bio-techne/Tocris, MN) and SCH79797 (Axon Medchem, VA), PLC inhibitor U73122 and its analog U73343 (Sigma, MO) were added to the cells and they were further incubated another 20 min.

Techniques: Isolation, Whisker Assay, Expressing, Concentration Assay, Flow Cytometry, Staining, Fluorescence

Journal: iScience

Article Title: The role of protease-activated receptor 1 signaling in CD8 T cell effector functions

doi: 10.1016/j.isci.2021.103387

Figure Lengend Snippet:

Article Snippet: After 30 min of incubation at 37°C and 5% CO 2 , media or the PAR1 inhibitors SCH530348 (Bio-techne/Tocris, MN) and SCH79797 (Axon Medchem, VA), PLC inhibitor U73122 and its analog U73343 (Sigma, MO) were added to the cells and they were further incubated another 20 min.

Techniques: Recombinant, Selection, Software

Journal: Heliyon

Article Title: The thrombin receptor (PAR1) is associated with microtubules, mitosis and process formation in glioma cells ☆

doi: 10.1016/j.heliyon.2024.e33329

Figure Lengend Snippet:

Article Snippet: Cells were seeded in 6-well plates at a density of 1 × 10 5 cells/well and allowed to attach for 24 h. The medium was then replaced by serum-free DMEM (3 ml) containing treatment (PAR1 antagonist SCH79797 (Tocris) [100 nM], thrombin (Sigma, T4648) [1U/ml]) for 3 h. Following the treatment, the cells were collected, and the RNA was extracted and cleaned according to the Bio-Rad Aurum 732–6820 kit instructions (Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Recombinant, Real-time Polymerase Chain Reaction, Software

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

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

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 4. Activated protein C (APC) binds to LA1 domain of soluble apolipoprotein E receptor 2 (ApoER2) on the reelin-binding surface. A, The binding of recombinant variants of sApoER2 containing LA domains 1, 2, 3, and 7 and epidermal growth factor A and B domains to immobilized APC was determined (Methods in the online-only Data Supplement). Binding data are presented as percentage of wild- type sApoER2 maximum binding that was defined as 100% based on the best fit of the binding data for a simple 1:1 association of wild- type sApoER2 to APC. Bars indicate SEM for data from 3 indepen- dent experiments, each with duplicate data points. B, LA1 domain CPK model based on crystal structure PDB ID 3A7Q29 is shown with previously identified surface areas for reelin binding indicated by yellow dashed-line oval and newly implicated APC binding surface indicated by black dashed-line oval. This ApoER2 LA1 domain model was prepared using Accelrys DS Viewer Pro software with residues numbered according to Yasui et al.29

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: Binding Assay, Recombinant, Software

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