Role of P2X, P2Y₂, P2Y₄, and A_2b receptors in HNP-induced IL-8 production. (A) A549 cells were incubated in serum-free DMEM and lipofectamine reagent preparation containing 2.5 μM P2Y₂ and P2Y₄ sense or antisense oligonucleotides, respectively, for 4 hours. RT-PCR of P2Y₂ mRNA and P2Y₄ mRNA analysis was performed. (B) Cells were incubated in serum-free DMEM and transfected with P2Y₂ and P2Y₄ sense or antisense oligonucleotides for 4 hours, washed with complete DMEM, and incubated overnight. After washing with PBS and incubation in serum-free DMEM containing 10 μg/mL HNP for 8 hours and IL-8, concentration was measured in supernatants (n = 3). (C) Cells were incubated in serum-free DMEM containing a final concentration of 10 μM PPADS or 30 μM alloxazine for 30 minutes, followed by the addition of 10 μg/mL HNP for 8 hours. IL-8 levels were measured in cell-culture supernatants (n = 3).