Fig 6.

SeV-induced but not TNF-α-induced nuclear translocation of endogenous NF-κB p65 is inhibited in LCMV-infected cells. (A) Nuclear translocation of endogenous NF-κB p65 in LCMV-infected cells. Subconfluent monolayers of A549 cells were mock infected (−LCMV) or infected (+LCMV, MOI = 10) with LCMV. At 20 h p.i., cells were mock infected (Mock) or infected with SeV (MOI = 3) and, at 3 h post-SeV infection, fixed, permeabilized, and immunostained with antibodies to endogenous NF-κB p65 (green) and LCMV-NP (red). Cellular nuclei were stained with DAPI (blue). Representative images are shown. (B) Percentages of NF-κB p65 nuclear translocation. Percentages of cells showing nuclear translocation of endogenous NF-κB p65 in mock-infected or LCMV-infected cells in response to SeV infection at the indicated hour postinfection were determined by counting 100 to 150 cells in nonoverlapping fields as described for Fig. 3C. (C) Nuclear translocation of endogenous NF-κB p65 in LCMV-infected cells upon TNF-α treatment. Subconfluent monolayers of A549 cells were mock infected (−LCMV) or infected with LCMV (+LCMV, MOI = 1) and 24 h later mock or TNF-α treated (50 ng/ml) for 4 h, fixed, permeabilized, and immunostained with a polyclonal antibody to endogenous NF-κB p65 and monoclonal antibody 1.1.3 to LCMV-NP. The percentage of cells showing nuclear translocation of endogenous NF-κB p65 in each sample was determined by counting 60 to 100 cells in nonoverlapping fields as described for Fig. 3C. (D) Levels of endogenous NF-κB p65 in cytosolic and nuclear lysates from mock- and LCMV-infected cells treated with TNF-α. Cytoplasmic (C) and nuclear (N) lysates of mock- and LCMV-infected cells treated with TNF-α (50 ng/ml) were analyzed by Western blotting using a polyclonal antibody for endogenous NF-κB p65. Caspase-3 (cytoplasmic) and poly-ADP ribose polymerase (PARP; nuclear) were used to determine purity. Mock-infected and TNF-α mock-treated cell extracts were used as a control.