FIG 2.
Effect of ectopically expressed NleF on NF-κB activity. (A) Immunofluorescence images of p65 localization using anti-N-terminal p65 (red) in HeLa cells 24 h after transfection with pRK5::myc-gfp, pRK5::myc-nleE, pRK5::myc-nleF, or pRK5::myc-nleF1–185 (green). Cells either were left unstimulated or were stimulated with 20 ng/ml TNF for 30 min, starting 24 h posttransfection. Cell nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Counts were taken for each condition of cells exhibiting either nuclear p65 or nuclear exclusion in 10 separate fields of view. A significant difference in p65 nuclear translocation was seen between GFP- and NleF-transfected cells and between GFP-transfected cells and untransfected cells (Un) stimulated with TNF. No statistical difference was reported between GFP- and NleE-transfected cells, between GFP- and NleF1–185-transfected cells, or between GFP-transfected cells and NleE-transfected cells stimulated with TNF. Results are averages for 3 independent repeats. Asterisks indicate significant differences (P < 0.05) from results for GFP-transfected cells. (C) Fold increase in NF-κB-dependent luciferase activity in cells transfected with pEGFP-C2 (GFP) or pGFP-NleF (NleF). NF-κB-dependent luciferase activity was significantly higher in NleF-transfected cells than in GFP-transfected cells. Results are means ± standard deviations for triplicate wells. Asterisks indicate significant differences (P < 0.05) from results for GFP-transfected cells.