FIG 7.

Effect of H₂S donor treatment on virus-induced signaling. (A and B) A549 cells were transiently transfected with an ISRE-driven (A) or NF-κB-driven (B) reporter gene plasmid, infected with RSV for 1 h, and then treated with 5 and 10 mM GYY4137. Cells were harvested at 15 or 24 h p.i. to measure luciferase and β-galactosidase reporter activities. Luciferase activity was normalized to the activity of the internal control β-galactosidase. Results are representative of data from three independent experiments run in triplicate. Data are expressed as means ± standard errors for normalized luciferase activity. *, P < 0.05 relative to untreated, RSV-infected cells. (C) A549 cells were infected with RSV for 1 h, followed by GYY4137 treatment at different concentrations, and harvested at 15 and 24 h p.i. to prepare either total cell lysates or nuclear extracts. IRF-3 and p65 nuclear translocation was assessed by Western blotting of nuclear extracts. Membranes were stripped and reprobed with lamin B to determine equal loading of the samples. (D) Total Ser276 and Ser536 p65 phosphorylation levels were determined by Western blotting of total cell lysates. The membrane was stripped and reprobed for total p65 and β-actin to determine equal loading of the samples. Data are representative of data from three independent experiments with similar results. (E) Chromatin DNA from uninfected and RSV-infected A549 cells in the presence or absence of GYY4137 for 15 h was immunoprecipitated by using an anti-NF-κB antibody (left), an anti-IRF-3 antibody (right), or IgG as a negative control. Q-gPCR was performed by using primers spanning either the NF-κB-binding site of the IL-8 promoter or the ISRE-binding site of the RANTES promoter. Total input chromatin DNA for immunoprecipitation was included as positive control for Q-gPCR amplification. The fold change was calculated compared to the IgG control. Results are representative of data from two independent experiments. *, P < 0.05 relative to untreated, RSV-infected cells.