Journal: International journal of molecular medicine

Article Title: Co-culture supernatants from Vibrio vulnificus-infected INT-407 cells induce IL-8 production in intestinal epithelial cells: crucial role of V. vulnificus rtxE.

doi: 10.3892/ijmm_00000510

Figure Lengend Snippet: Figure 6. Effects of co-culture supernatants from rtxE CT V. vulnificus-infected INT-407 cells on NF-κB DNA activation and IκB· phosphorylation. (A) Human intestinal epithelial cells were transiently co-transfected with the NF-κB minimal promoter/luciferase construct and pRL-TK reporter vector, followed by 12 or 24 h of treatment with co-culture supernatants from WT, rtxE MT, or rtxE CT V. vulnificus-infected INT-407 cells. Afterwards, the cells were washed with PBS and the luciferase activity was determined. The results were normalized by Renilla luciferase activity, and are expressed as the relative fold induction. The data are representative of 3 independent experiments. The data are expressed as the means ± standard errors (n=3). *P<0.01, relative to the group treated with co-culture supernatants from WT V. vulnificus-infected INT-407 cells at 24 h. **P<0.05, relative to the group treated with co-culture supernatants from rtxE MT V. vulnificus-infected INT-407 cells at 24 h. (B) Human intestinal epithelial cells were treated for 1-3 h with co-culture supernatants from WT, rtxE MT, or rtxE CT V. vulnificus-infected INT-407 cells. NF-κB DNA binding activity in the nuclear extracts was assessed via EMSA. S (specific) and NS (non-specific) indicate the presence of a 50-fold excess of specific oligonucleotide (NF-κB) and non-specific oligonucleotide (CRE-containing oligonucleotide), respectively. (C) Human intestinal epithelial cells were treated with co-culture supernatants from WT, rtxE MT, or rtxE CT V. vulnificus-infected INT-407 cells. The cell lysates were prepared and analyzed by Western blot analysis using anti-IκB·, anti-pIκB· and ß-actin antibodies. The data are representative of 3 independent experiments. (D) Reduced NF-κB p65 nuclear translocation in human intestinal epithelial cells treated with co- culture supernatants from rtxE MT V. vulnificus-infected INT-407 cells. Human intestinal epithelial cells grown in 24-well plates were treated for 3 h with co- culture supernatants from WT, rtxE MT, or rtxE CT V. vulnificus-infected INT-407 cells. The cells were washed, fixed, permeabilized, and stained with anti- p65 polyclonal antibody followed by staining with FITC-conjugated secondary antibody (green) or rhodamine phalloidin (red; actin staining). NF-κB p65-stained cells were merged with rhodamine phalloidin actin staining. The data are representative of 3 independent experiments. WT, wild-type; rtxE MT, rtxE mutant; rtxE CT, rtxE-complemented.

Article Snippet: The cells were incubated with a rabbit antiNF-κB p65 antibody (diluted 1:200) for 1 h at room temperature, and then incubated with fluorescein isothiocyanateconjugated secondary antibody (Santa Cruz Biotechnology).

Techniques: Co-Culture Assay, Infection, Activation Assay, Phospho-proteomics, Transfection, Luciferase, Construct, Plasmid Preparation, Activity Assay, Binding Assay, Western Blot, Translocation Assay, Staining, Mutagenesis