Activation of NF-κB.

<p>(A) A549 cells were transfected with either pORF3-EGFP (ORF3) or pEGFP-N1 (GFP) for 48 h, stimulated with TNF-α (50 ng/ml) for 6 h, and then subjected to western blotting, using the untreated cells as a control (Contr). (B) Immunofluorescence laser confocal microscopy for the detection of p65. A549 cells were transfected with either pORF3-EGFP (ORF3) or pEGFP-N1 (GFP) for 48 h and then stimulated with TNF-α (50 ng/ml) for 6 h, with another untreated group regarded as the control. The distribution of p65 was examined using p65-specific monoclonal antibody (red). The nucleus was counterstained with DAPI and observed under a confocal laser scanning microscope (magnification: 100×). (C) TNF-α induced NF-κB DNA-binding activity in pORF3-expressing cells. After 6 h of TNF-α treatment, the nuclear extracts were used to determine the NF-κB activity by EMSA. NC, negative control; PC, positive control; ORF3, transfected with pORF3; GFP, transfected with empty pEGFP-N1; UN, untreated cells; CA, competition assay; MCA, mutant competitive assay. (D) Effects of pORF3 on TNF-α-induced NF-κB target genes by real-time RT-PCR. The relative expression of IL-1β, COX-2, and ICAM-1 was calculated following normalization to β-actin, and fold changes relative to the expression levels in untreated cells (control) are presented. (E) The levels of IL-1β, COX-2, and ICAM-1 secreted in the supernatant of the culture medium were detected by ELISA. The protein values used for the calculation were obtained from a corresponding standard curve. The data are presented as mean ± SD from three independent experiments (each performed in triplicate). *P<0.05, ΔP>0.05.