f2rl3 par4  (Addgene inc)

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) sCD13 showed significant correlations with Cit-H3 ( n = 172) and MPO-DNA ( n = 172) in patients with COVID-19 while S100 A8/A9 ( n = 172) showed a positive slope but not a statistically significant correlation. ( B ) Representative images of neutrophils isolated from peripheral blood and analyzed after stimulation with PBS or sCD13. Panels show merged images of NETs in which neutrophil elastase was stained green by immunofluorescence and DNA was stained blue by Hoechst 33342. n = 3 technical replicates. Scale bar: 100 μm. ( C ) sCD13 blocked the staining of PAR4-5F10 antibodies, which recognize only the inactivated/uncleaved form of PAR4 yet had no effect on PAR4-FITC antibodies, which recognize both the activated and inactivated forms. n = 3 technical replicates, repeated 2 times. Original magnification, ×1,000. ( D ) sCD13-induced NETosis was blocked by B1R inhibitor SSR-240612 and PAR4 inhibitor BMS-986120 (all n = 3). ( E ) PMA-induced NETosis was not impacted by the B1R or PAR4 inhibitors (all n = 3). Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Significance was determined by Spearman’s test ( A ) and 1-way ANOVA ( D and E ).

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Isolation, Staining, Immunofluorescence

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: Single-cell RNA-Seq results of nasopharyngeal/pharyngeal swabs, bronchial brushings and bronchial lavages were generated from patients with COVID-19 ( n = 19) and healthy controls ( n = 5). Data are extracted from Chua et al. . ( A ) Both ANPEP (codes for CD13) and MMP14 are expressed on various epithelial cells and macrophages. ANPEP is also expressed on neutrophils while MMP14 is expressed on mast cells. BDKRB1 (codes for B1R) is expressed on some epithelial cells with the highest expression on secretory cells and ciliated cells. F2RL3 (codes for PAR4) was barely detected in this dataset but seemed to be expressed by epithelial cells. Cell abbreviations are defined in . ( B ) Cellular ANPEP expression in nasopharyngeal cells is elevated in patients with COVID-19 (moderate COVID-19 n = 8, severe COVID-19 n = 11) compared with healthy controls ( n = 5). ( C ) Using the median expression levels of ANPEP in patients, patients with COVID-19 were divided into 2 populations: ANPEP -high and ANPEP -low. Differentially expressed genes in neutrophils from these 2 groups were shown in the volcano plot ( P < 1 × 10 –20 , |log 2 (fold-change)| < 0.25). ( D ) Pathway analysis of the differentially expressed genes in ANPEP -high and ANPEP -low neutrophils showed pathways related to neutrophil degranulation, leukocyte activation, and inflammation. ( E ) The neutrophils from ANPEP -high patients with COVID-19 ( n = 10) showed a gene signature of immature-like neutrophils characterized by the overexpression of genes coding for several granule-content proteins (healthy controls n = 5, ANPEP -low n = 9). ( F ) The score for neutrophil immaturity was higher in critically ill patients with COVID-19 ( n = 11) compared with moderate patients (left, n = 8). The median neutrophil-immaturity score of neutrophils in ANPEP -high patients ( n = 10) was lower than that in ANPEP -low patients (right, n = 9). ( G ) The neutrophil-immaturity signature was most prominent in ANPEP -high patients ( n = 10) as developing neutrophils versus mature neutrophils from the peripheral blood were examined (healthy controls n = 5, ANPEP -low n = 9). Results are expressed as mean ± SD. **** P < 0.0001. Significance was determined by 1-way ANOVA ( B and G ) and Mann-Whitney test ( F ).

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Single Cell, RNA Sequencing, Generated, Expressing, Analysis, Activation Assay, Over Expression, MANN-WHITNEY, Single Cell RNA-Seq

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) Gating scheme of flow cytometry analysis on monocytes and neutrophils. ( B ) CD13, B1R, and PAR4 were expressed on monocytes and neutrophils, while MMP14 showed minimal expression on these cells ( n = 3). ( C ) Histograms showing the changes in CD13, PAR4, and B1R expression in neutrophils and monocytes with or without IL-1β, TNF-α, or IL-6 stimulation. ( D ) Quantification of relative expression of CD13, PAR4, and B1R on neutrophils and monocytes after stimulation with IL-1β, TNF-α, or IL-6 compared with unstimulated control from 3 healthy donors. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. Significance was determined by 1-way ANOVA. FMO, fluorescence minus 1 (gating control); NT, not treated.

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Flow Cytometry, Analysis, Expressing, Control, Fluorescence, FACS

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) Reanalysis of single-cell RNA-Seq results generated from circulating human neutrophils revealed that immature neutrophils have higher ANPEP expression. Dot plot of ANPEP , IFN, and neutrophil maturity genes for each neutrophil cluster, showing the average expression level and the percentage of cells expressing the gene in each cluster. ( B ) Gating scheme of flow cytometry analysis on mature and immature neutrophils isolated from whole blood. CD10 + CD16 hi defines mature neutrophils while CD10 – CD16 lo defines immature neutrophils. ( C ) Significantly higher expression of CD13 and lower expression of PAR4 were observed in mature neutrophils compared with immature neutrophils while B1R expression showed similar levels. Data generated from 5–10 healthy controls. Results are expressed as mean ± SD. ** P < 0.01. Significance was determined by Wilcoxon’s test.

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Single Cell, RNA Sequencing, Generated, Expressing, Flow Cytometry, Analysis, Isolation, FACS

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: Antibodies to TP (27159-1-AP), P2Y1 (67654-1-AP), and PAR4 (25306-1-AP) were purchased from Proteintech Group, Inc (Rosemont, IL).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: To investigate how α1 affects platelet GPCR signaling function and its interaction with certain platelet GPCRs, we transfected COS-7 cells (ATCC, CRL-1651) with plasmids encoding P2Y12 (item#66471, Addgene), F2RL3 (PAR4) (item #66279), TP (item #66517), or A2BR (item#37202) using LipofectamineTM 3000 Transfection Reagent (L3000015, ThermoFisher Scientific).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control