Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 1. RT-PCR analysis of human leu- kocytes and endothelial cells for PAR RNA expression. Total RNA was ex- tracted from human eosinophils (A), neu- trophils (B), mononuclear cells (C), or en- dothelial cells (HUVECs; D) and was sub- ject to RT-PCR using random hexamer primers for the RT step and PAR-specific primers for the PCR step. PCR products were analyzed by agarose gel electro- phoresis and compared with a 100-bp lad- der (left lane). Expected product sizes were: PAR1, 300 bp; PAR2, 399 bp; PAR3, 372 bp; PAR4, 242 bp. Figure is a representative example of five different donors.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Random Hexamer, Agarose Gel Electrophoresis

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 3. Flow cytometry of human eosinophils for PAR1 and PAR2. Purified human eosinophils were stained with mAb to PAR1 (ATAP2, A and B) or PAR2 (SAM11, C and D). Cells were unfixed to study surface-only expression (A and C) or fixed and permeabilized to look at surface and intracellular levels (B and D). mAb are shown by the solid line, and appropriate isotype controls are represented by the dotted lines. (A–D) Representative example of four independent experiments. The levels of PAR1 and PAR2 in purified eosinophils or mixed granulocyte population following fixation and permeabilization were measured in control and asthmatic donors (each group, n4). (E) The results are shown as a ratio of the mean fluorescence intensity (MFI) of PAR mAb:isotype control.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Flow Cytometry, Staining, Expressing, Control

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 5. GAFS assay to measure shape change in response to PAR agonists in eosinophils (Eos) and neutrophils (Neuts). The GAFS assay was used to assess shape change by measuring increases in FSc of a mixed population of eosinophils and neu- trophils using flow cytometry. The eosinophil pop- ulation (R1) is distinguished from the neutrophils (R2) by their autofluorescent properties on the FL2 channel (A). Resting eosinophils and neutrophils (C and D) showed an increase in mean FSc when stimulated with PAF (E, P0.001; F, P0.001). (B) The mean FSc of resting and stimulated cells from a typical experiment is shown. Eotaxin was also used as a control to increase the mean FSc in eosinophils only. (G and H) The results shown are expressed as the percent increase in FSc induced by each agonist compared with buffer-stimulated cells after 5 and 30 min incubation (n6). Eosin- ophils (G) responded to the PAR1 peptide at both time points (5 min, P0.004; 30 min, P0.010) but not to thrombin. Activation via trypsin or PAR2 peptide (P0.008) was only seen after 30 min. Neutrophils (H) responded to trypsin after 30 min (P0.033) and at 5 and 30 min with peptides to PAR1 (5 min, P0.023; 30 min, P0.009) and PAR2 (5 min, P0.021; 30 min, P0.001). PAR4 was considered ineffective in this assay in either cell type.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Cytometry, Control, Incubation, Activation Assay

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 6. Release of cys-LT from human eosinophils in response to PAR activation. Purified human eosinophils were primed with 5 ng/ml IL-5 and treated with PAR agonists for 60 min. Cell-free supernatants were assayed for cys-LT using a commercially available EIA (n4–7). Release of cys-LT was very donor-variable, and only PAR2 peptide stimulated significant release compared with assay buffer alone (P0.0189). As a positive control, eosino- phils were treated with IgG-coupled sepharose beads, and release of over 4 ng/ml cys-LT was detected (data not shown).

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Activation Assay, Positive Control

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 7. Generation of ROS in human eosinophils in response to PAR activa- tion. Eosinophils were resuspended in assay buffer containing lucigenin and stimulated with PAR ligands and peptides. ROS generation was measured by chemiluminescence, and 1 M PAF was used as a positive control (P0.0041). Thrombin was inactive, whereas trypsin (P0.0062) or the PAR2 peptide (P0.0107) induced significant ROS generation. The PAR2 control peptide also stimulated some ROS generation, but this was significantly reduced compared with the active peptide (P0.0175).

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Positive Control, Control

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 expression level in lung cancer cells transfected either with pcDNA3-PAR2 or PAR2 shRNA. (A) PAR2 expression level after A549 cells were transfected with pcDNA3-PAR2; (B) PAR2 expression level after H1299 cells were transfected with pcDNA3-PAR2; (C) PAR2 expression level after A549 cells were transfected with PAR2 shRNA; (D) PAR2 expression level after H1299 cells were transfected with PAR2 shRNA.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, shRNA

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: Overexpression PAR2 promoted growth of lung cancer cells with or without paclitaxel. (A–D) The growth of A549 cells with or without paclitaxel after transfection with pcDNA3-PAR2; (E–H) The growth of H1299 cells with or without paclitaxel after transfection with pcDNA3-PAR2.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Transfection

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: Knockdown PAR2 decreased growth of lung cancer cells with or without paclitaxel. (A–D) The growth of A549 cells with or without paclitaxel after transfection with PAR2 shRNA; (E–H) The growth of H1299 cells with or without paclitaxel after transfection with PAR2 shRNA.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Transfection, shRNA

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 inhibited paclitaxel-associated apoptosis in lung cancer cells. (A) Caspase 3/7 activity in A549 cell transfected with pcDNA3-PAR2; (B) Caspase 3/7 activity in H1299 cell transfected with pcDNA3-PAR2; (C–F) Bcl-2 and BAX expression in A549 and H1299 cells.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Transfection, Expressing

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 played essential roles in invasion and migration of lung cancer cells. (A, B) up-regulation of PAR2 increased migration and invasion of A549 cells. (C, D) down-regulation of PAR2 decreaased migration and invasion of A549 cells. (E, F) up-regulation of PAR2 increased migration and invasion of H1299 cells. (G, H) down-regulation of PAR2 decreased migration and invasion of H1299 cells.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Migration

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 altered PTEN/AKT protein expression in lung cancer cells. (A–C) Impact of up-regulation of PAR2 on expression of p-AKT, AKT and PTEN in A549 and H1299 cells. (D–F) Impact of down-regulation of PAR2 on expression of p-AKT, AKT and PTEN in A549 and H1299 cells.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 expression in human lung cancer tissue. (A) PAR2 levels in different stage of lung cancer tissue and normal lung tissue. (B) Immunohistochemical studies for Ki-67 and PTEN on different levels of PAR2 in lung tissue.

Article Snippet: PAR2 shRNA lentiviral particles are purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Immunohistochemical staining

Journal: Communications biology

Article Title: The PAR2 inhibitor I-287 selectively targets Gα q and Gα 12/13 signaling and has anti-inflammatory effects.

doi: 10.1038/s42003-020-01453-8

Figure Lengend Snippet: Fig. 5 I-287 inhibits PAR2-mediated activation of DAG/Ca2+/PKC and RhoA/SRF-RE, as well as FAK and ERK1/2 signaling pathways. a, b Impact of increasing concentrations of I-287 (15 min) on DAG production (a) and PKC activation (b) induced after 1 (DAG) or 5 (PKC) min stimulation with an EC80 concentration of hTrypsin or SLIGKV-NH2 in HEK293 cells co-expressing hPAR2 and the indicated unimolecular BRET2-based biosensors. Results are expressed as ΔBRET in % of the response induced by EC80 of respective agonists in the absence of I-287 (mean ± SEM; n = 4–5). c Impact of increasing concentrations of I-287 (30 min) on intracellular Ca2+ mobilization induced by an EC80 concentration of hTrypsin or SLIGKV-NH2 in HEK293 cells endogenously expressing hPAR2. Results are expressed as % of the response induced by respective agonists in the absence of I-287 (mean ± SEM; n = 3-4). d Impact of I-287 (10 µM, 30 min) on hPAR2-promoted SRF-RE reporter gene activation induced after 6 h stimulation with hTrypsin (10 U/mL) or SLIGKV-NH2 (100 µM) in HEK293 cells expressing hPAR2. FBS (10%) was used as control. Results are expressed as % of the response induced by respective agonists in the absence of I-287 (mean ± SEM; n = 3–5; unpaired t-test: *p < 0.05 and **p < 0.01 compared to respective control cells, ns: nonsignificant). e, f Kinetics of FAK and ERK1/2 phosphorylation in HEK293 cells expressing hPAR2 and pretreated with DMSO or I-287 (10 µM, 30 min) before stimulation with hTrypsin (1 U/mL) or SLIGKV-NH2 (100 µM) at the indicated times. Representative immunoblots of FAK and ERK1/2 phosphorylation are shown. Western blots were quantified and expressed as the ratio of phosphorylated protein level (P-FAK or P-ERK1/2) normalized over total protein (t-FAK or t-ERK1/2; mean ± SEM; n = 3–5; two-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to DMSO-treated cells at the respective time).

Article Snippet: Mouse PAR2 (mPAR2) cDNA plasmid was purchased from R&D Systems (catalog number RDC0167) and cloned between the BamHI and XbaI sites of pCDNA3.1/Zeo(+) vector.

Techniques: Activation Assay, Protein-Protein interactions, Concentration Assay, Expressing, Control, Phospho-proteomics, Western Blot

Journal: Communications biology

Article Title: The PAR2 inhibitor I-287 selectively targets Gα q and Gα 12/13 signaling and has anti-inflammatory effects.

doi: 10.1038/s42003-020-01453-8

Figure Lengend Snippet: Fig. 8 Effect of I-287 on intracellular signaling pathways induced by the two human PAR2 agonists, Trypsin, and SLIGKV-NH2. The pathways inhibited by I-287 are in black, whereas the unaffected pathways are in gray.

Article Snippet: Mouse PAR2 (mPAR2) cDNA plasmid was purchased from R&D Systems (catalog number RDC0167) and cloned between the BamHI and XbaI sites of pCDNA3.1/Zeo(+) vector.

Techniques: Protein-Protein interactions

Journal: Communications biology

Article Title: The PAR2 inhibitor I-287 selectively targets Gα q and Gα 12/13 signaling and has anti-inflammatory effects.

doi: 10.1038/s42003-020-01453-8

Figure Lengend Snippet: Fig. 5 I-287 inhibits PAR2-mediated activation of DAG/Ca2+/PKC and RhoA/SRF-RE, as well as FAK and ERK1/2 signaling pathways. a, b Impact of increasing concentrations of I-287 (15 min) on DAG production (a) and PKC activation (b) induced after 1 (DAG) or 5 (PKC) min stimulation with an EC80 concentration of hTrypsin or SLIGKV-NH2 in HEK293 cells co-expressing hPAR2 and the indicated unimolecular BRET2-based biosensors. Results are expressed as ΔBRET in % of the response induced by EC80 of respective agonists in the absence of I-287 (mean ± SEM; n = 4–5). c Impact of increasing concentrations of I-287 (30 min) on intracellular Ca2+ mobilization induced by an EC80 concentration of hTrypsin or SLIGKV-NH2 in HEK293 cells endogenously expressing hPAR2. Results are expressed as % of the response induced by respective agonists in the absence of I-287 (mean ± SEM; n = 3-4). d Impact of I-287 (10 µM, 30 min) on hPAR2-promoted SRF-RE reporter gene activation induced after 6 h stimulation with hTrypsin (10 U/mL) or SLIGKV-NH2 (100 µM) in HEK293 cells expressing hPAR2. FBS (10%) was used as control. Results are expressed as % of the response induced by respective agonists in the absence of I-287 (mean ± SEM; n = 3–5; unpaired t-test: *p < 0.05 and **p < 0.01 compared to respective control cells, ns: nonsignificant). e, f Kinetics of FAK and ERK1/2 phosphorylation in HEK293 cells expressing hPAR2 and pretreated with DMSO or I-287 (10 µM, 30 min) before stimulation with hTrypsin (1 U/mL) or SLIGKV-NH2 (100 µM) at the indicated times. Representative immunoblots of FAK and ERK1/2 phosphorylation are shown. Western blots were quantified and expressed as the ratio of phosphorylated protein level (P-FAK or P-ERK1/2) normalized over total protein (t-FAK or t-ERK1/2; mean ± SEM; n = 3–5; two-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to DMSO-treated cells at the respective time).

Article Snippet: Plasmids. hPAR2 cDNA plasmid was purchased from Origen (IBD90.1 clone; catalog number SC322345), where the serine in position 291 has been mutated in threonine (S291T) to reproduce the hPAR2 phenotype observed in HEK293 and HCT 116 cells.

Techniques: Activation Assay, Protein-Protein interactions, Concentration Assay, Expressing, Control, Phospho-proteomics, Western Blot