p2x7 antagonist  (MedChemExpress)

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: The vasoconstrictor adenosine 5'-tetraphosphate is a danger signal that induces IL-1β.

doi: 10.1186/s10020-025-01116-6

Figure Lengend Snippet: Fig. 3 Effects of Ap4 on P2X7 receptor. A and B HEK293 cells were transfected with P2X7 receptor (pUNO1-hP2RX7) or control plasmid (pUNO-mcs). A After transfection, HEK293 cells were loaded with Fluo-4 AM and probenecid. Ap4 (5 mM), BzATP (300 µM) or calcimycin (10 µM) were added after 20 s. Calcium influx was determined by fluorescence intensity measurement. The graph shows the mean values related to the basal level (n = 3). B Cells were loaded with YO-PRO-1 (2 µM) and stimulated for 1 h without or with BzATP (300 µM) or Ap4 (5 mM). Flow cytometry was used to quantify the change in YO-PRO-1 uptake, as indicated by fluorescence intensity in the FITC channel, after stimulation with either Ap4 or BzATP (depicted by colored curves), in comparison to the untreated control (represented by light grey curves). Heat-killed cells were used as positive control. The dark grey curve shows unstained cells. The histograms are representative of three independent experiments. Mean + SEM (n = 3–6). C THP-1 macrophages were primed with Pam3CSK4 (1 µg/ml) for 3 h and then stimulated with BzATP (300 µM) for 3 h. Ap4 (5 mM) was added 1 h before stimulation. IL-1β release in cell culture supernatants was determined by ELISA. IL-1β release induced by BzATP was set to 100%. All other values were calculated accordingly. Bar graphs show mean ± SEM (n = 3). One-sample t-test against 100%, *P ≤ 0.05

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM; 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP) (300 μM; 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD [5-(3-Bromophenyl)1,3-dihydro-2 H-Benzofuro[3,2-e]-1,4-diazepin-2-one] (25 μM; SML0450-5MG, Sigma-Aldrich, Taufkirchen, Germany), P2 receptor antagonist PPADS (100 μM; 0625, Tocris Bioscience, Bristol, United Kingdom), Gq/11 inhibitor YM-254,890 (1 μM; AG-CN2-0509, Biomol, Hamburg, Germany), NLRP3 inhibitor MCC950 (10 μM; 5479, Tocris Bioscience, Bristol, United Kingdom) or NF-κB inhibitor Bay 11-7082 (20 μM; B5556, SigmaAldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM; 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VAD-fmk (40 μM; tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM; 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM; 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM; 2936, Carl Roth, Karlsruhe, Germany), l-homocysteine (100 μM; Cay30852-5, Biomol, Hamburg, Germany), or ion chelator TPEN [N, N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine] (10 μM; Cay13340-10, Biomol, Hamburg, Germany) for 1 h before stimulation.

Techniques: Transfection, Control, Plasmid Preparation, Fluorescence, Flow Cytometry, Comparison, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay