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(A) Western blot analysis of NFAT1, NFAT2, NFAT3, and NFAT4 was performed on lysates from murine platelets or the uterine tissue. One experiment representative of three experiments. (B) Phos-tag western blot analysis of mouse platelets pretreated, or not, with myristoylated VIVIT (MyrVIVIT) (10 μM) for 30 min and then stimulated with <t>PAR4-AP</t> (100 μM) for the indicated time points. One experiment representative of three experiments. (C) Murine platelets were pretreated, or not, with vehicle (DMSO), the myristoylated control peptide (MyrVEET), or MyrVIVIT (10 μM) for 10 min and then activated with PAR4-AP (75 μM). Aggregation was monitored for 5 min. Histograms represent the percentage of maximal aggregation and area under curve (AUC). Data represent mean ± SEM. n ≥ 3 independent experiments. (D) Human platelets were treated as in (C) and stimulated with TRAP (5 μM). Histograms represent the percentage of the maximal aggregation and AUC. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Platelet-rich plasma (PRP) from WT or Nfat1−/− mice was stimulated with PAR4-AP (150 μM). Histograms represent the percentage of maximal aggregation and AUC. Each dot represents an independent test and lines highlight measurements performed concomitantly. (F) PRP from WT or Nfat1−/− mice was stimulated with PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation was analyzed by cytofluorimetry. Bars represent the mean fluorescence intensity (MFI). Data represent mean ± SEM. n ≥ 3 independent experiments. (G) Representative phos-tag western blot analysis of murine platelets pretreated, or not, with 1 μM FK506 for 1 h and then stimulated with PAR4-AP (100 μM) for the indicated time. (H) Murine PRP was treated as in (G). Aggregation curves and histograms of maximal aggregation and AUC are shown. Each dot represents an independent test. (I) PRP was pretreated as in (G) and then stimulated with different concentration of PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation was analyzed by cytofluorimetry. Bars represent the MFI. Data represent mean ± SEM. n ≥ 3 independent experiments. (J) PRP from WT or Nfat1−/− mice was pretreated as in (G), and integrin αIIbβ3 activation was analyzed by cytofluorimetry. Data represent mean ± SEM. n ≥ 3 independent experiments. One-way ANOVA (C and D), unpaired two-tailed t test (E and H), or two-way ANOVA (F, I, and J) were used for statistics. n.s., not significant (p > 0.05), **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S1.

Par4 Ap, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia"

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

Journal: Immunity

doi: 10.1016/j.immuni.2021.12.002

Figure Legend Snippet: (A) Western blot analysis of NFAT1, NFAT2, NFAT3, and NFAT4 was performed on lysates from murine platelets or the uterine tissue. One experiment representative of three experiments. (B) Phos-tag western blot analysis of mouse platelets pretreated, or not, with myristoylated VIVIT (MyrVIVIT) (10 μM) for 30 min and then stimulated with PAR4-AP (100 μM) for the indicated time points. One experiment representative of three experiments. (C) Murine platelets were pretreated, or not, with vehicle (DMSO), the myristoylated control peptide (MyrVEET), or MyrVIVIT (10 μM) for 10 min and then activated with PAR4-AP (75 μM). Aggregation was monitored for 5 min. Histograms represent the percentage of maximal aggregation and area under curve (AUC). Data represent mean ± SEM. n ≥ 3 independent experiments. (D) Human platelets were treated as in (C) and stimulated with TRAP (5 μM). Histograms represent the percentage of the maximal aggregation and AUC. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Platelet-rich plasma (PRP) from WT or Nfat1−/− mice was stimulated with PAR4-AP (150 μM). Histograms represent the percentage of maximal aggregation and AUC. Each dot represents an independent test and lines highlight measurements performed concomitantly. (F) PRP from WT or Nfat1−/− mice was stimulated with PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation was analyzed by cytofluorimetry. Bars represent the mean fluorescence intensity (MFI). Data represent mean ± SEM. n ≥ 3 independent experiments. (G) Representative phos-tag western blot analysis of murine platelets pretreated, or not, with 1 μM FK506 for 1 h and then stimulated with PAR4-AP (100 μM) for the indicated time. (H) Murine PRP was treated as in (G). Aggregation curves and histograms of maximal aggregation and AUC are shown. Each dot represents an independent test. (I) PRP was pretreated as in (G) and then stimulated with different concentration of PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation was analyzed by cytofluorimetry. Bars represent the MFI. Data represent mean ± SEM. n ≥ 3 independent experiments. (J) PRP from WT or Nfat1−/− mice was pretreated as in (G), and integrin αIIbβ3 activation was analyzed by cytofluorimetry. Data represent mean ± SEM. n ≥ 3 independent experiments. One-way ANOVA (C and D), unpaired two-tailed t test (E and H), or two-way ANOVA (F, I, and J) were used for statistics. n.s., not significant (p > 0.05), **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S1.

Techniques Used: Western Blot, Control, Activation Assay, Fluorescence, Concentration Assay, Two Tailed Test

(A) PRP from mice treated, or not, with 4 mg/kg of FK506 for 14 days were stimulated with PAR4-AP (150 μM). Maximal aggregation and AUC are shown. Each dot represents an independent test and lines highlight measurements performed concomitantly. (B) Tail bleeding test was performed on mice treated, or not, with FK506 (left panel) or on WT and Nfat1−/− mice (right panel). Each dot represents a mouse (n = 29 for FK-treated mice, n = 16 for Nfat1−/− mice). (C) Mice treated, or not, with 4 mg/kg of FK506 for 14 days (left) or WT and Nfat1−/− mice (right) were intravenously injected with a mixture of collagen (500 μg/kg) and epinephrine (3 μg/kg) and were scored for a pathology index. Each dot represents a mouse (n = 9 for FK-treated mice, n = 5 for Nfat1−/− mice). (D) Tail bleeding test (left panel) and thromboembolism test (right panel) were performed on HPS mice treated, or not, with FK506. Each dot represents a mouse (n = 6). Unpaired two-tailed t test was used for statistics. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S3.

Figure Legend Snippet: (A) PRP from mice treated, or not, with 4 mg/kg of FK506 for 14 days were stimulated with PAR4-AP (150 μM). Maximal aggregation and AUC are shown. Each dot represents an independent test and lines highlight measurements performed concomitantly. (B) Tail bleeding test was performed on mice treated, or not, with FK506 (left panel) or on WT and Nfat1−/− mice (right panel). Each dot represents a mouse (n = 29 for FK-treated mice, n = 16 for Nfat1−/− mice). (C) Mice treated, or not, with 4 mg/kg of FK506 for 14 days (left) or WT and Nfat1−/− mice (right) were intravenously injected with a mixture of collagen (500 μg/kg) and epinephrine (3 μg/kg) and were scored for a pathology index. Each dot represents a mouse (n = 9 for FK-treated mice, n = 5 for Nfat1−/− mice). (D) Tail bleeding test (left panel) and thromboembolism test (right panel) were performed on HPS mice treated, or not, with FK506. Each dot represents a mouse (n = 6). Unpaired two-tailed t test was used for statistics. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S3.

Techniques Used: Injection, Two Tailed Test

(A) Whole blood from mice treated, or not, with 4 mg/kg of FK506 for 14 days was stimulated, or not, with PAR4-AP (100 μM). The formation of complexes between platelets and neutrophils was analyzed by cytofluorimetry. Results show the MFI for the platelet marker CD41 on neutrophils, identified as Ly6G+CD11b+ cells. Data represent mean ± SEM. n ≥ 3 independent experiments. (B) Platelet-neutrophil complex formation was analyzed by ImageStream. Platelets treated, or not, with 1 μM FK506 for 1 h and identified with different fluorochromes, were washed, mixed with the blood from platelet-depleted mice, and stimulated (PAR4-AP), or not (Unstim), with PAR4-AP (25 μM). The percentage of neutrophils (identified as Ly6G+ cells in the whole blood) that did not bind any platelets (0), or that bound 1 or 2 platelets (1–2), or 3 or more platelets (>3) was measured for both platelets pretreated (FK-506) or not (NT) with FK506. Data represent mean ± SEM. n ≥ 3 independent experiments. (C) PRP was pretreated, or not, with 1 μM FK506 for 1 h and stimulated with PAR4-AP (150 μM) for 5 min. PAR4-AP-activated platelets were then used to induce NETosis. Histograms represent the percentage of neutrophils positive for the citrullinated histone H3 (left). Each dot represents an independent test. A representative image of NET released by neutrophils is shown (right). Scale bars, 10 μm. (D) Murine PRP was pretreated, or not, with 1 μM FK506 for 1 h and then stimulated with different concentration of PAR4-AP (150 and 100 μM). P-selectin exposure was analyzed by cytofluorimetry and P-selectin MFI is shown. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Neutrophils were stimulated as in (C) in the presence of the anti-P-selectin antibody or respective isotype control. Data represent mean ± SEM. n ≥ 3 independent experiments. One-way ANOVA (A, D, and E), two-way ANOVA (B), or unpaired two-tailed t test (C) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. See also Figure S4.

Figure Legend Snippet: (A) Whole blood from mice treated, or not, with 4 mg/kg of FK506 for 14 days was stimulated, or not, with PAR4-AP (100 μM). The formation of complexes between platelets and neutrophils was analyzed by cytofluorimetry. Results show the MFI for the platelet marker CD41 on neutrophils, identified as Ly6G+CD11b+ cells. Data represent mean ± SEM. n ≥ 3 independent experiments. (B) Platelet-neutrophil complex formation was analyzed by ImageStream. Platelets treated, or not, with 1 μM FK506 for 1 h and identified with different fluorochromes, were washed, mixed with the blood from platelet-depleted mice, and stimulated (PAR4-AP), or not (Unstim), with PAR4-AP (25 μM). The percentage of neutrophils (identified as Ly6G+ cells in the whole blood) that did not bind any platelets (0), or that bound 1 or 2 platelets (1–2), or 3 or more platelets (>3) was measured for both platelets pretreated (FK-506) or not (NT) with FK506. Data represent mean ± SEM. n ≥ 3 independent experiments. (C) PRP was pretreated, or not, with 1 μM FK506 for 1 h and stimulated with PAR4-AP (150 μM) for 5 min. PAR4-AP-activated platelets were then used to induce NETosis. Histograms represent the percentage of neutrophils positive for the citrullinated histone H3 (left). Each dot represents an independent test. A representative image of NET released by neutrophils is shown (right). Scale bars, 10 μm. (D) Murine PRP was pretreated, or not, with 1 μM FK506 for 1 h and then stimulated with different concentration of PAR4-AP (150 and 100 μM). P-selectin exposure was analyzed by cytofluorimetry and P-selectin MFI is shown. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Neutrophils were stimulated as in (C) in the presence of the anti-P-selectin antibody or respective isotype control. Data represent mean ± SEM. n ≥ 3 independent experiments. One-way ANOVA (A, D, and E), two-way ANOVA (B), or unpaired two-tailed t test (C) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. See also Figure S4.

Techniques Used: Marker, Concentration Assay, Control, Two Tailed Test

(A) PRP was pretreated, or not, with 1 μM FK506 for 1 h and incubated, or not, for 10 min with 3 μM ARC-66096 (ARC) then stimulated with different concentration of PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation and P-selectin exposure were analyzed by cytofluorimetry. Bars represent the MFI. Data represent mean ± SEM. n ≥ 3 independent experiments. (B) PRP was pretreated, or not, with 1 μM FK506 for 1 h and the release of ATP was measured upon stimulation with PAR4-AP (150 μM). Histogram represents maximal ATP release. Data represent mean ± SEM. n ≥ 3 independent experiments. (C and D) PRP was pretreated as in (B) and then stimulated with ADP (20 μM). Aggregation (C) and integrin αIIbβ3 activation and P-selectin exposure were analyzed (D). Data represent mean ± SEM. n ≥ 3 independent experiments. (E) PRP was pretreated as in (B) and then stimulated with PAR4-AP (150 μM) alone or in combination with ADP (10 nM). Integrin αIIbβ3 activation and P-selectin exposure were analyzed by cytofluorimetry. Data show mean ± SEM. n ≥ 3 independent experiments. Two-way ANOVA (A, D, and E) or unpaired two-tailed t test (B) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. See also Figure S5.

Figure Legend Snippet: (A) PRP was pretreated, or not, with 1 μM FK506 for 1 h and incubated, or not, for 10 min with 3 μM ARC-66096 (ARC) then stimulated with different concentration of PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation and P-selectin exposure were analyzed by cytofluorimetry. Bars represent the MFI. Data represent mean ± SEM. n ≥ 3 independent experiments. (B) PRP was pretreated, or not, with 1 μM FK506 for 1 h and the release of ATP was measured upon stimulation with PAR4-AP (150 μM). Histogram represents maximal ATP release. Data represent mean ± SEM. n ≥ 3 independent experiments. (C and D) PRP was pretreated as in (B) and then stimulated with ADP (20 μM). Aggregation (C) and integrin αIIbβ3 activation and P-selectin exposure were analyzed (D). Data represent mean ± SEM. n ≥ 3 independent experiments. (E) PRP was pretreated as in (B) and then stimulated with PAR4-AP (150 μM) alone or in combination with ADP (10 nM). Integrin αIIbβ3 activation and P-selectin exposure were analyzed by cytofluorimetry. Data show mean ± SEM. n ≥ 3 independent experiments. Two-way ANOVA (A, D, and E) or unpaired two-tailed t test (B) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. See also Figure S5.

Techniques Used: Incubation, Concentration Assay, Activation Assay, Two Tailed Test

(A) Schematic of the iNFATuation transgenic mouse model. (B) Blood from WT, littermates or iNFATuation-Pf4-cre mice was stained for the platelet marker CD41 and analyzed for expression of VIVIT-tdTomato signal. One experiment representative of three experiments. (C) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM). Aggregation was monitored for 5 min. Histograms of maximal aggregation and AUC are shown. Each dot represents an independent test. (D) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM). P-selectin exposure was analyzed by cytofluorimetry, and P-selectin MFI is shown. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Whole blood from littermates or iNFATuation-Pf4-cre mice was treated with PAR4-AP (100 μM). The formation of complexes between platelets and neutrophils was analyzed by cytofluorimetry. Results show the MFI for the platelet marker CD41 on neutrophils, identified as Ly6G+CD11b+ cells. Data represent mean ± SEM. n ≥ 3 independent experiments. (F) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM) for 5 min. PAR4-AP-activated platelets were then used to induce NETosis. Histograms represent the percentage of neutrophils positive for the citrullinated histone H3. Each dot represents an independent test. (G) Tail bleeding test (left panel) and thromboembolism test (right panel) were performed on littermates or iNFATuation-Pf4-cre mice. Each dot represents a mouse (n = 4 littermates, n = 6 iNFATuation-Pf4-cre). Data show mean ± SEM. Unpaired two-tailed t test (C, F, and G) and two-way ANOVA (D and E) were used for statistics. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S6.

Figure Legend Snippet: (A) Schematic of the iNFATuation transgenic mouse model. (B) Blood from WT, littermates or iNFATuation-Pf4-cre mice was stained for the platelet marker CD41 and analyzed for expression of VIVIT-tdTomato signal. One experiment representative of three experiments. (C) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM). Aggregation was monitored for 5 min. Histograms of maximal aggregation and AUC are shown. Each dot represents an independent test. (D) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM). P-selectin exposure was analyzed by cytofluorimetry, and P-selectin MFI is shown. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Whole blood from littermates or iNFATuation-Pf4-cre mice was treated with PAR4-AP (100 μM). The formation of complexes between platelets and neutrophils was analyzed by cytofluorimetry. Results show the MFI for the platelet marker CD41 on neutrophils, identified as Ly6G+CD11b+ cells. Data represent mean ± SEM. n ≥ 3 independent experiments. (F) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM) for 5 min. PAR4-AP-activated platelets were then used to induce NETosis. Histograms represent the percentage of neutrophils positive for the citrullinated histone H3. Each dot represents an independent test. (G) Tail bleeding test (left panel) and thromboembolism test (right panel) were performed on littermates or iNFATuation-Pf4-cre mice. Each dot represents a mouse (n = 4 littermates, n = 6 iNFATuation-Pf4-cre). Data show mean ± SEM. Unpaired two-tailed t test (C, F, and G) and two-way ANOVA (D and E) were used for statistics. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S6.

Techniques Used: Transgenic Assay, Staining, Marker, Expressing, Two Tailed Test

Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Virus, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software, Luminex

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Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: (A) Western blot analysis of NFAT1, NFAT2, NFAT3, and NFAT4 was performed on lysates from murine platelets or the uterine tissue. One experiment representative of three experiments. (B) Phos-tag western blot analysis of mouse platelets pretreated, or not, with myristoylated VIVIT (MyrVIVIT) (10 μM) for 30 min and then stimulated with PAR4-AP (100 μM) for the indicated time points. One experiment representative of three experiments. (C) Murine platelets were pretreated, or not, with vehicle (DMSO), the myristoylated control peptide (MyrVEET), or MyrVIVIT (10 μM) for 10 min and then activated with PAR4-AP (75 μM). Aggregation was monitored for 5 min. Histograms represent the percentage of maximal aggregation and area under curve (AUC). Data represent mean ± SEM. n ≥ 3 independent experiments. (D) Human platelets were treated as in (C) and stimulated with TRAP (5 μM). Histograms represent the percentage of the maximal aggregation and AUC. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Platelet-rich plasma (PRP) from WT or Nfat1−/− mice was stimulated with PAR4-AP (150 μM). Histograms represent the percentage of maximal aggregation and AUC. Each dot represents an independent test and lines highlight measurements performed concomitantly. (F) PRP from WT or Nfat1−/− mice was stimulated with PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation was analyzed by cytofluorimetry. Bars represent the mean fluorescence intensity (MFI). Data represent mean ± SEM. n ≥ 3 independent experiments. (G) Representative phos-tag western blot analysis of murine platelets pretreated, or not, with 1 μM FK506 for 1 h and then stimulated with PAR4-AP (100 μM) for the indicated time. (H) Murine PRP was treated as in (G). Aggregation curves and histograms of maximal aggregation and AUC are shown. Each dot represents an independent test. (I) PRP was pretreated as in (G) and then stimulated with different concentration of PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation was analyzed by cytofluorimetry. Bars represent the MFI. Data represent mean ± SEM. n ≥ 3 independent experiments. (J) PRP from WT or Nfat1−/− mice was pretreated as in (G), and integrin αIIbβ3 activation was analyzed by cytofluorimetry. Data represent mean ± SEM. n ≥ 3 independent experiments. One-way ANOVA (C and D), unpaired two-tailed t test (E and H), or two-way ANOVA (F, I, and J) were used for statistics. n.s., not significant (p > 0.05), **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S1.

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Western Blot, Control, Activation Assay, Fluorescence, Concentration Assay, Two Tailed Test

Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: (A) PRP from mice treated, or not, with 4 mg/kg of FK506 for 14 days were stimulated with PAR4-AP (150 μM). Maximal aggregation and AUC are shown. Each dot represents an independent test and lines highlight measurements performed concomitantly. (B) Tail bleeding test was performed on mice treated, or not, with FK506 (left panel) or on WT and Nfat1−/− mice (right panel). Each dot represents a mouse (n = 29 for FK-treated mice, n = 16 for Nfat1−/− mice). (C) Mice treated, or not, with 4 mg/kg of FK506 for 14 days (left) or WT and Nfat1−/− mice (right) were intravenously injected with a mixture of collagen (500 μg/kg) and epinephrine (3 μg/kg) and were scored for a pathology index. Each dot represents a mouse (n = 9 for FK-treated mice, n = 5 for Nfat1−/− mice). (D) Tail bleeding test (left panel) and thromboembolism test (right panel) were performed on HPS mice treated, or not, with FK506. Each dot represents a mouse (n = 6). Unpaired two-tailed t test was used for statistics. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S3.

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Injection, Two Tailed Test

Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: (A) Whole blood from mice treated, or not, with 4 mg/kg of FK506 for 14 days was stimulated, or not, with PAR4-AP (100 μM). The formation of complexes between platelets and neutrophils was analyzed by cytofluorimetry. Results show the MFI for the platelet marker CD41 on neutrophils, identified as Ly6G+CD11b+ cells. Data represent mean ± SEM. n ≥ 3 independent experiments. (B) Platelet-neutrophil complex formation was analyzed by ImageStream. Platelets treated, or not, with 1 μM FK506 for 1 h and identified with different fluorochromes, were washed, mixed with the blood from platelet-depleted mice, and stimulated (PAR4-AP), or not (Unstim), with PAR4-AP (25 μM). The percentage of neutrophils (identified as Ly6G+ cells in the whole blood) that did not bind any platelets (0), or that bound 1 or 2 platelets (1–2), or 3 or more platelets (>3) was measured for both platelets pretreated (FK-506) or not (NT) with FK506. Data represent mean ± SEM. n ≥ 3 independent experiments. (C) PRP was pretreated, or not, with 1 μM FK506 for 1 h and stimulated with PAR4-AP (150 μM) for 5 min. PAR4-AP-activated platelets were then used to induce NETosis. Histograms represent the percentage of neutrophils positive for the citrullinated histone H3 (left). Each dot represents an independent test. A representative image of NET released by neutrophils is shown (right). Scale bars, 10 μm. (D) Murine PRP was pretreated, or not, with 1 μM FK506 for 1 h and then stimulated with different concentration of PAR4-AP (150 and 100 μM). P-selectin exposure was analyzed by cytofluorimetry and P-selectin MFI is shown. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Neutrophils were stimulated as in (C) in the presence of the anti-P-selectin antibody or respective isotype control. Data represent mean ± SEM. n ≥ 3 independent experiments. One-way ANOVA (A, D, and E), two-way ANOVA (B), or unpaired two-tailed t test (C) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. See also Figure S4.

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Marker, Concentration Assay, Control, Two Tailed Test

Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: (A) PRP was pretreated, or not, with 1 μM FK506 for 1 h and incubated, or not, for 10 min with 3 μM ARC-66096 (ARC) then stimulated with different concentration of PAR4-AP (150 and 100 μM). Integrin αIIbβ3 activation and P-selectin exposure were analyzed by cytofluorimetry. Bars represent the MFI. Data represent mean ± SEM. n ≥ 3 independent experiments. (B) PRP was pretreated, or not, with 1 μM FK506 for 1 h and the release of ATP was measured upon stimulation with PAR4-AP (150 μM). Histogram represents maximal ATP release. Data represent mean ± SEM. n ≥ 3 independent experiments. (C and D) PRP was pretreated as in (B) and then stimulated with ADP (20 μM). Aggregation (C) and integrin αIIbβ3 activation and P-selectin exposure were analyzed (D). Data represent mean ± SEM. n ≥ 3 independent experiments. (E) PRP was pretreated as in (B) and then stimulated with PAR4-AP (150 μM) alone or in combination with ADP (10 nM). Integrin αIIbβ3 activation and P-selectin exposure were analyzed by cytofluorimetry. Data show mean ± SEM. n ≥ 3 independent experiments. Two-way ANOVA (A, D, and E) or unpaired two-tailed t test (B) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, and ****p < 0.0001. See also Figure S5.

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Incubation, Concentration Assay, Activation Assay, Two Tailed Test

Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: (A) Schematic of the iNFATuation transgenic mouse model. (B) Blood from WT, littermates or iNFATuation-Pf4-cre mice was stained for the platelet marker CD41 and analyzed for expression of VIVIT-tdTomato signal. One experiment representative of three experiments. (C) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM). Aggregation was monitored for 5 min. Histograms of maximal aggregation and AUC are shown. Each dot represents an independent test. (D) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM). P-selectin exposure was analyzed by cytofluorimetry, and P-selectin MFI is shown. Data represent mean ± SEM. n ≥ 3 independent experiments. (E) Whole blood from littermates or iNFATuation-Pf4-cre mice was treated with PAR4-AP (100 μM). The formation of complexes between platelets and neutrophils was analyzed by cytofluorimetry. Results show the MFI for the platelet marker CD41 on neutrophils, identified as Ly6G+CD11b+ cells. Data represent mean ± SEM. n ≥ 3 independent experiments. (F) PRP from littermates or iNFATuation-Pf4-cre mice was stimulated with PAR4-AP (150 μM) for 5 min. PAR4-AP-activated platelets were then used to induce NETosis. Histograms represent the percentage of neutrophils positive for the citrullinated histone H3. Each dot represents an independent test. (G) Tail bleeding test (left panel) and thromboembolism test (right panel) were performed on littermates or iNFATuation-Pf4-cre mice. Each dot represents a mouse (n = 4 littermates, n = 6 iNFATuation-Pf4-cre). Data show mean ± SEM. Unpaired two-tailed t test (C, F, and G) and two-way ANOVA (D and E) were used for statistics. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S6.

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Transgenic Assay, Staining, Marker, Expressing, Two Tailed Test

Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Virus, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software, Luminex

Journal: Immunity

Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

doi: 10.1016/j.immuni.2021.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PAR4-AP , MedChemExpress , Cat# HY-P1309.

Techniques: Virus, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software, Luminex

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