Effects of P2Y receptor antagonists suramin and reactive blue HNP-induced IL-8 production, and HNP binding on A549 cells. (A) A549 cells (2.5 × 10⁵ cells/well in 6-well plate) were incubated in serum-free DMEM containing 100 μM suramin or reactive blue for 30 minutes, followed by the addition of 10 μg/mL HNP for 8 hours. IL-8 levels in supernatants were measured. ^*P < .05 suramin or reactive blue compared with no inhibitor, respectively. (B) A549 cells (5 × 10⁴ cell/well in 96-well plate) were incubated in serum-free DMEM containing 0 to 0.7 μM ¹²⁵I-labeled HNP for 30 minutes on ice, with or without pretreatment with 40-fold molar excess concentration of unlabeled HNP for 30 minutes. Cell-associated HNP binding was measured by radioactivity counting. CPM indicates counts per minute. (C) HNPs do not compete with ATP or UDP in engaging cell-surface binding sites. A549 cells were incubated in serum-free DMEM containing 0 to 2.8 μM HNP for 30 minutes, with or without pretreatment with 0 to 100 μM ATP or UDP for 30 minutes. A control group of cells incubated with UDP in the absence of HNP was included. Cell-surface HNP binding was measured by HNP-cell ELISA (n = 3).