Figure 6.

P2Y2 purinergic receptor antagonist pretreatment of Raw264.7 macrophages inhibits macrophage-mediated induction of iNOS and apoptosis in AML12 hepatocytes in vitro. Raw264.7 macrophages were treated with LPS (100 ng/mL) for 7.5 h or pretreated with Vehicle (Veh, 0.2% DMSO) or AR-C (50 µM, P2Y2 purinergic receptor antagonist) for 30 min before LPS treatment. AML-12 hepatocytes were treated with macrophage culture supernatant (conditioned media) for 30 h, and total protein extracts of AML-12 hepatocytes were analyzed by Western blotting with antibodies (A and B) specific for iNOS and β-actin (protein loading control; n, Veh, 3; AR-C, 3; Veh+LPS, 3; AR-C+LPS, 3). C–E: antibodies specific for cleaved PARP-1, cleaved Caspase-3, and β-actin [n, Untreated (Un), 3; Veh, 3; Veh +LPS, 3; AR-C+LPS, 3]. Bar diagrams represent relative expression. Means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n, number of biological replicates.