Salmonella infection leads to NF-κB nuclear localization even in non-infected cells. HT29 cells were grown on glass coverslips and either mock-infected, left untreated, infected with Salmonella typhimurium, or treated with TNFα (10 ng/ml). Cells fixed after 30 min (TNF) and 1 h (Salmonella) as described in Experimental Procedures and Salmonella that had invaded HT29 cells were detected by direct fluorescence microscopy of GFP expression, p65(RelA) localization was monitored by indirect immunoflourescence of rabbit anti-p65 antibody detected with FITC-conjugated donkey anti-rabbit antibody. DAPI was used to stain nuclei. A, HT29 cells were mock-infected or infected at an MOI of 50 with Salmonella typhimurium strain SJW1103G which expresses GFP from the ssaH promoter that is only active inside infected host cells [10,34]. Cells were photographed using bright field microscopy (BF), and immunoflourescence to detect GFP or DAPI staining as indicated. Images were merged (overlay) to reveal cells that were infected. B, HT29 cells were left untreated, infected with Salmonella typhimurium strain 1103 or treated with TNFα. NF-κB p65(RelA) localization under various conditions as indicated was monitored by indirect immunofluorescence. Cells were visualized by bright field microscopy (BF), cell nuclei were stained with DAPI and p65(RelA) was visualized with FITC. DAPI staining was falsely colored red to make visualization of the merge (overlay) easier to distinguish.