Fig 2: eAdo inhibits Nlrp3 inflammasome and this occurs in A2B-cAMP-NRF2--HO-1 dependent manner. Panel (A) Effect of eAdo on eATP mediated activation in vitro of Nlrp3 inflammasome in presence or absence A2A and A2B inhibitors evaluated by Caspase-Glo® 1 Inflammasome assay (Promega). BM MNC were isolated, and caspase 1 activation was measured. Experiments were repeated three times. ns – not significant, **p < 0.001. Panel (B) The effect of Ado, Ado plus A2B inhibitor PSB1115 or A2A inhibitor SCH442416 (10 µM of each) on the phosphorylation of the intracellular the nuclear factor (NF)-kappa B p65(RelA)ser536 intracellular pathway protein in treated murine bone marrow derived mononuclear cells was analyzed by western blotting. These cells (2 × 106 cells per ml) were rendered quiescent for 3 h in RPMI medium containing 0.5% BSA at 37 °C, and afterwards the protein lysates were collected after 5 min of stimulation. Proteins retrieved from cells cultured in only assay medium were served as a control. Panel (C) Western blotting for expression of nuclear factor-like 2 (Nrf2) in protein lysates (40 µg of each sample) collected from pre-treated mononuclear cells. After incubation of cells with the target compounds at mentioned above concentrations, protein was immediately extracted and then quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and Multimode Analysis Software (Beckman Coulter Inc, Fullerton CA, USA). Expression of ß-actin was also analyzed to ensure that all lanes were equivalently loaded. Panel (D) Effect of adenosine and its receptor antagonists on intracellular levels of cyclic AMP in pre-treated MNCs (4 × 104 cells for each sample). cAMP concentrations were detected by cyclic AMP Assay kit (Cell signaling; #4339). *P < 0.05 is considered statistically significant between pre-treated and untreated cells. Panel (E) Expression of HO-1 in protein lysates collected from pre-treated mononuclear cells with adenosine with or without A2B (PSB1115) or A2A (SCH442416) inhibitor at the mentioned above concentrations for 1 h at 37oC. ß-actin was also analyzed to ensure that the lanes were equivalently loaded. Panel (F) RT-qPCR analysis of murine HO-1 transcripts in mRNA samples that were purified from mouse MNCs, cultured with the tested compounds in a serum-free medium for 1 h at 37 °C. ß2-microglobulin was used as an endogenous control. Samples containing only water instead of cDNA were used in each run as a negative control. Cells without any stimulation served as a control. *P < 0.05 is considered statistically significant between pre-treated and untreated cells