Journal: Nature Communications

Article Title: Neutrophil P2X 7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP

doi: 10.1038/ncomms10555

Figure Lengend Snippet: ( a ). Western blot analysis of P2X 7 receptors on unstimulated and LPS-primed bone marrow neutrophils and macrophages from C57BL/6 and P2X 7 −/− mice. ( b – e ). Flow cytometry and fluorescence microscopy of P2X 7 R ecto domain (using the Hano-3 antibody) in LPS-primed bone marrow neutrophils that were either permeabilized with 0.1% TX-100 to detect total P2X 7 R ( b , c ) or were non-permeabilized to detect only cell surface P2X 7 R ( d , e ). ( c,e ) Cells were also stained with DAPI to detect the nucleus; Scale bar, 5 μm). ( f – h ). Representative profiles of ATP-induced Ca 2+ influx in bone marrow neutrophils from C57BL/6 and P2X 7 −/− mice ( f ), and in C57BL/6 neutrophils after stimulation with ATP (3 mM) in the presence of P2X 7 R antagonists AZ10606120 (10 μM) ( g ), or with different concentrations of A438079 ( h ). ( i – k ). Mean±s.e.m. pf Area under the curve (AUC) of intracellular Ca 2+ from three independent experiments. A P value ≤0.05 was considered significant using an unpaired Student's t- test.

Article Snippet: P2X 7 R inhibitors AZ10606120 dihydrochloride and AZ11645373, A438079 hydrochloride (Tocris Bioscience) were dissolved in DMSO and used at concentrations noted in the results.

Techniques: Western Blot, Flow Cytometry, Fluorescence, Microscopy, Staining

Journal: Nature Communications

Article Title: Neutrophil P2X 7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP

doi: 10.1038/ncomms10555

Figure Lengend Snippet: Total IL-1β secreted by bone marrow neutrophils from C57BL/6 and P2X 7 −/− mice ( a , b ), or from C57BL/6 neutrophils incubated with the P2X 7 antagonists AZ10606120 (10 μM) ( c ) or A438079 (25 μM) ( d ) following LPS priming and stimulation with ATP or nigericin measured by ELISA. ( e – g ). Bioactive IL-1 from the same samples detected using HEK-Blue-IL-1R reporter cells. ( h ). IL-1α and IL-1β from the C57BL/6 neutrophil supernatants stimulated 45 min with 3 mM ATP and 10 μM nigericin were quantified by ELISA. Data points are mean±s.d. of at least three replicates per treatment and are representative of three independent experiments. Using an ANOVA with Tukey post hoc analysis, a P value ≤0.05 was considered significant.

Article Snippet: P2X 7 R inhibitors AZ10606120 dihydrochloride and AZ11645373, A438079 hydrochloride (Tocris Bioscience) were dissolved in DMSO and used at concentrations noted in the results.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: Vitexin Alleviates Kainic Acid-Induced Seizure Through Inhibiting P2X7R/NLRP3 Signaling Pathway

doi: 10.1007/s10753-025-02324-2

Figure Lengend Snippet: P2X7R contributes to VT’s antiseizure and neuroprotective effects. A Relative protein expression of P2X7R. n = 6, F 2, 15 = 12.19. B Relative mRNA level of P2X7R. n = 6, F 2, 15 = 16.68. C Schematic representation of the experimental timeline. D Seizure scores of A438079 pre-treatment in KA mice. n = 10, F 2, 69 = 575.5. E Average seizure scores of A438079 pre-treatment in KA mice. n = 10, t = 23.15. F Effects of A438079 pre-treatment on the latency to seizure onset in KA-induced seizure model. n = 10, t = 6.185. G The generalized seizure duration after A438079 pre-treatment in KA mice. n = 10, t = 21.63. H Effects of A438079 pre-treatment on the mortality in KA mice. n = 10. I Typical immunohistochemical images of Nissl staining. Scale bars = 100–500 μm. J Representative images of H.E. staining. Scale bars = 100–500 μm. K Effects of A438079 pre-treatment on the Nissl positive neurons in CA3 area in KA mice. n = 3, F 2, 6 = 29.17. L Effects of A438079 pre-treatment on neuronal damage in CA3 area in KA mice. n = 3, F 2, 6 = 19.95. Data are shown as Means ± SEM. ** P < 0.01, *** P < 0.001. One-way ANOVA with Tukey’s post-hoc test was used in A, B, D, K and L. Unpaired two-tailed Student’s t-test was used in E-G

Article Snippet: Valproate (VPA) (HY-10585 A), A438079 (HY-15488), and BzATP (HY-136254) were obtained from Med Chem Express (MCE, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Inflammation

Article Title: Vitexin Alleviates Kainic Acid-Induced Seizure Through Inhibiting P2X7R/NLRP3 Signaling Pathway

doi: 10.1007/s10753-025-02324-2

Figure Lengend Snippet: A438079 suppresses NLRP3 inflammasome activation in KA mice. A Representative immunoreactive bands of hippocampal NLRP3, P2X7R, IL-1β, ASC, Caspase-1. B - F Relative protein level of hippocampal P2X7R (B, n = 6, F 2, 15 = 7.961), NLRP3 (C, n = 6, F 2, 15 = 73.08), ASC (D, n = 6, F 2, 15 = 108.0), Caspase-1 (E, n = 6, F 2, 14 = 7.611) and IL-1β (F, n = 6, F 2, 15 = 6.961), respectively. G - K Relative mRNA levels of hippocampal P2X7R (G, n = 3, F 2, 6 = 9.168), NLRP3 (H, n = 3, F 2, 6 = 28.99), ASC (I, n = 3, F 2, 6 = 18.26), Caspase-1 (J, n = 3, F 2, 6 = 7.693) and IL-1β (K, n = 3, F 2, 6 = 7.716), respectively. * P < 0.05, ** P < 0.01. Data are expressed as means ± SEM. One-way ANOVA with Tukey’s post-hoc test

Article Snippet: Valproate (VPA) (HY-10585 A), A438079 (HY-15488), and BzATP (HY-136254) were obtained from Med Chem Express (MCE, USA).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Extracellular ATP Limits Homeostatic T Cell Migration Within Lymph Nodes

doi: 10.3389/fimmu.2021.786595

Figure Lengend Snippet: ATP inhibits CCL19-induced chemotaxis but not S1P-induced chemotaxis of CD4 + T cells and CD8 + T cells via P2X7R. (A) CCL19-dependent T cell chemotaxis in the presence or absence of ATP was analyzed by Transwell assay. The result shown is representative of two independent experiments. Data represent the mean ± SD of triplicate wells. * p <0.05 by Student’s t -test. (B) The effect of ATP on CCL19-dependent T cell chemotaxis in the presence of ATP or apyrase-treated ATP in the lower well. The result shown is representative of three independent experiments. Data represent the mean ± SD of triplicate wells. * p <0.05 by Student’s t -test. (C) T cell chemotaxis in the presence of ATP and P2 receptor antagonists. CD4 + and CD8 + T cells were treated with A-438079 (P2X7R inhibitor; 10 μM), NF023 (P2X1R inhibitor; 10 μM), or 5-BDBD (P2X4R inhibitor; 10 μM) and subjected to the CCL19-dependent chemotaxis assay in the presence or absence of 100 μM ATP in the lower well. The result shown is representative of two independent experiments. Data represent the mean ± SD of triplicate wells. * p <0.05 by Student’s t -test. (D) Expression of P2X7R on CD3 + CD49d low T cells in LNs analyzed by flow cytometry. P2X7R is shown in black. Isotype control is shown in grey. The result shown is representative of at least three independent experiments. (E) An apparent contents map of ATP (ATP app ) generated as previously described and a confocal microscopic image of a serial LN section stained for CD4 (red), PNAd (green), and nucleus (blue). Images are representative of three individual experiments. (F) The effect of the P2X7R antagonist A-438079 on the intranodal retention of circulating lymphocytes. Data represent the mean ± SEM of five mice. * p <0.05 by Student’s t -test. (G) The effect of ATP on S1P-dependent T cell chemotaxis. S1P or CCL21 was added to the lower wells in the presence or absence of ATP at the indicated concentrations, and T cells were added to the upper wells. Relative cell migration was determined as described above. The result shown is representative of two independent experiments. Data represent the mean ± SD of triplicate wells. * p <0.05 by Student’s t -test; NS, not significant.

Article Snippet: In the P2X antagonist experiments, serum-starved LN cells were treated with 10 μM of A-438079 (Wako Pure Chemical), NF023 (Tocris Bioscience), or 5-BDBD (Tocris Bioscience) for 30 min at 37°C ( ) before adding these cells to the upper chamber.

Techniques: Chemotaxis Assay, Transwell Assay, Expressing, Flow Cytometry, Generated, Staining, Migration