Fig. 3

Activation of PAR-1 and -2, and the effects of PAR-1 antagonist and siRNA knockdown of PAR-1 and PAR-2 on the induction of IL-8 mRNA and IL-8 protein levels. a A549 cells were transduced with adenovirus expressing recombinant PAR-1 or PAR-2 containing alkaline phosphatase linked at the amino terminus, and treated with medium (C), dust extract (DE), or DE in the presence of protease inhibitor cocktail (PIC) and alkaline phosphatase activity in the medium was measured by chemiluminiscent assay. Data shown are means of duplicate measurements. Similar results were obtained in two other independent experiments. b A549 cells were first incubated with medium (C) or PAR-1 antagonist BMS200261 (50 μM) for 1 h and then treated with dust extract (DE) (0.25 %) for 3 h. IL-8 levels in the medium were measured by ELISA. Data shown are means ± SE (n = 4). *P < 0.05, #P < 0.0001. c and d A549 cells were transfected with control siRNA (C), PAR-1, PAR-2, or a combination of both siRNAs (PAR1 + PAR2) and after 60 h treated with medium or dust extract (0.25 %) (DE) for 3 h. IL-8 mRNA levels and secreted IL-8 protein levels in cell medium were determined by qRT-PCR and ELISA, respectively. IL-8 mRNA and protein levels in dust extract treated cells transfected with control siRNA (C) were arbitrarily set as 100 and relative levels in PAR-1 and PAR-2 siRNA transfected cells are shown. Data are means ± SE (n = 3). *P < 0.05; ***P < 0.001; #P < 0.0001 compared with cells transfected with control siRNA and treated with dust extract