Fig 3. Effect of inactivated-H5N1 is mediated via P2Y₆ purinoceptors.

The effect of inactivated-H5N1 on IL-6 (A and C) and CXCL8 (B and D) mRNA expression. 16HBE14o- cells were incubated with vehicle (dimethyl sulfoxide (DMSO) or H₂O as appropriate) as control, 1 U/ml sialidase, 2 μM cytochalasin D (cytoD) (A and B) or with apyrase (2 U/ml), suramin (100 μM), MRS2578 (10 μM) or MRS2179 (20 μM) (C and D), for 1 hr before exposure to inactivated-H5N1. Three hours after treatment, cells were harvested for qRT-PCR. Data were normalized with corresponding control groups from the same batch of cells treated with inactivated-H5N1 (open bars) and reported as a percentage. (A and B) cells were treated (+) or untreated (-) with inactivated-H5N1 as indicated. (E) 16HBE14o- cells were transfected with siRNA directed against P2Y₆R or a scrambled-siRNA (100 pmol). Forty-eight hours after transfection, cells were exposed to inactivated-H5N1 for 3 hr before being harvested and the mRNA expression of P2Y₆R analyzed. (F) Cells in (E) were incubated with inactivated-H5N1 for 3 hr before being harvested and analyzed for mRNA expression of IL-6 and CXCL8. Data were normalized with a group of cells treated with allantoic fluid, as described, and reported as a percentage of normalized scrambled-siRNA treated group. Values are means ± SEM from at least 3 sets of experiments. *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively (unpaired Student’s t-test).