Fig 4. The cellular signaling mechanism by which inactivated-H5N1 increases mRNA for IL-6 and CXCL8 involves intracellular Ca²⁺, PLC, PKC, ERK1/2 and p38 MAPK.

16HBE14o- cells were incubated with BAPTA-AM (50 μM), U73122 (10 μM), BIM (1 μM), Gö6976 (10 μM) (A and B), or with SN-50 (10 μM), PD98059 (50 μM), SB203580 (10 μM) or SP600125 (10 μM) (C and D), for 1 hr before being exposed to inactivated-H5N1. Three hours later, mRNA expression of IL-6 (A and C) or that of CXCL8 (B and D) was analyzed. All data were normalized with corresponding control groups treated with inactivated-H5N1 (open bars) and reported as a percentage. (E) Cells were treated with SB203580 (10 μM) for 1 hr before incubation with inactivated-H5N1 (20 μg/ml hemagglutinin). Thirty-six hours later, the cell-free culture medium was harvested and analyzed for the presence of IL-6 and CXCL8 proteins as described in Fig 1. Data are presented as a percentage of control (treated with inactivated-H5N1, open bar). Values are means μ SEM from at least 3 sets of experiments. *, ** and *** indicates p < 0.05, p < 0.01 and p < 0.001 compared with control, respectively (unpaired Student’s t-test).