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

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: A central role for nuclear factor-κB pathway in the antiinflammatory and proinflammatory actions of mechanical strain

doi: 10.1096/fj.02-0901fje

Figure Lengend Snippet: A) Electrophoretic mobility shift assay (EMSA) analysis of nuclear extracts of untreated cells or cells treated with IL-1β (1 ng/ml), TENS-L (6%), or TENS-L (6%) and IL-1β for 30, 60, 90, or 180 min, showing inhibition of NF-κB nuclear translocation by TENS-L. B) Quantitative analysis of 32P associated with each band shown in A. C) Analysis of subunits of NF-κB involved in TENS-L actions by supershift EMSA, using antibodies to p65 (Rel A) and p50 subunits of NF-κB. D) Nuclear translocation of NF-κB as assessed by immunofluroscence. NF-κB was stained with anti-NF-κB p65 primary IgG and TRITC-conjugated donkey anti-rabbit IgG as second antibodies (white arrows). Cellular β-actin is shown by FITC-conjugated phalloidin (red arrows). The panels show that in untreated control cells, NF-κB is cytoplasmic at all time points. In cells treated with rhIL-1β (1 ng/ml) for 30, 60, 120, or 180 min, NF-κB completely translocated to the nucleus, with some cytoplasmic NF-κB at 60, 120, and 180 min. In cells treated with TENS alone, NF-κB was localized in the cytoplasm. In cells treated with TENS and rhIL-1β for 30, 60, 120, and 180 min, TENS-L (6%) markedly abrogated IL-1β-induced nuclear translocation of NF-κB at all time points as evidenced by its presence in the cytoplasm. E) Inhibition of IL-1β-induced I-κBβ degradation by TENS-L (6%) by immunofluorescence analysis. Panels show the presence of cytoplasmic I-κBβ in untreated control cells and cells treated with TENS-L (6%) alone (white arrows) over a period of 30–120 min. Cells treated with IL-1β exhibit near complete absence of I-κBβ due to its degradation within the first 30 min and its resynthesis at 60 and 120 min. Cells treated with TENS-L and IL-1β show the suppression of IL-1β-induced I-κBβ degradation by TENS-L as evidenced by the presence of I-κBβ in the cytoplasm at all time points tested. Cellular actin was stained by phalloidin-FITC to reveal the cell morphology (red arrows).

Article Snippet: In some experiments, we analyzed the nuclear translocation of NF-κB by immunofluorescence, using rabbit antiNF-κB p65 IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and CY3 conjugated goat anti-rabbit IgG (Jackson Laboratories, Bar Harbor, ME).

Techniques: Electrophoretic Mobility Shift Assay, Inhibition, Translocation Assay, Staining, Control, Immunofluorescence

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: A central role for nuclear factor-κB pathway in the antiinflammatory and proinflammatory actions of mechanical strain

doi: 10.1096/fj.02-0901fje

Figure Lengend Snippet: A) Cells were either untreated or treated with TENS-H (15%) in the presence or absence of IL-1β (1 ng/ml) and subjected to immunofluorescence staining as described in Figure 4D. Panels show the presence of NF-κB in the cytoplasms in untreated control cells (white arrows). Exposure of periodontal ligament (PDL) cells to 15% TENS-H, IL-1β, or TENS-H and IL-1β for 30–120 min resulted in rapid and sustained nuclear translocation of the majority of NF-κB as evidenced by red stained nuclei (white arrows). Actin was stained with FITC Phalloidin to show the morphology of PDL cells. B) Quantitative assessment of bands obtained by electrophoretic mobility shift assay (EMSA) analysis of nuclear extracts of untreated cells or cells treated with IL-1β (0.4 ng/ml), TENS-H (15%), or TENS-H and IL-1β for 30, 60, 90, or 180 min, demonstrating NF-κB nuclear translocation by TENS-H and IL-1β and the additive effects of TENS-H and IL-1β. C) Subunit structure of NF-κB translocated to the nucleus following exposure of cells to TENS-H by supershift EMSA, using antibodies to p65 (Rel A) and p50 subunits of NF-κB. D) Inhibition of TENS-H-induced nuclear translocation of NF-κB by caffeic acid phenylethylester (CAPE). PDL cells were either untreated or treated with CAPE (25 μM) for 10 min and then exposed to TENS (15%) for 30, 60, 120, or 180 min. The inhibition of TENS-H-induced nuclear translocation of NF-κB by CAPE was analyzed by quantitative assessment of each band in gels following EMSA analysis. E) Inhibition of TENS-H-induced COX-2 mRNA expression in cells exposed to CAPE. PDL cells were exposed to various concentrations of CAPE (0, 5, 10, 15, 25, or 50 μM) for 10 min and subsequently exposed to TENS-H for 4 h. RT-PCR analysis showing that TENS-H induced COX-2 mRNA expression was completely abrogated by 25 μM CAPE. F) TENS-H (15%) induced degradation of I-κBβ in PDL cells. Western blot analysis showing the presence of I-κBβ in the cytoplasmic fraction of untreated control cells and cells treated with neutralized IL-1β (1 ng/ml + 2 μg of rabbit anti-IL-1β IgG), whereas 30-min exposure of cells to TENS-H, IL-1β (1 ng/ml), or TENS-H and IL-1β resulted in a rapid degradation of I-κBβ.

Article Snippet: In some experiments, we analyzed the nuclear translocation of NF-κB by immunofluorescence, using rabbit antiNF-κB p65 IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and CY3 conjugated goat anti-rabbit IgG (Jackson Laboratories, Bar Harbor, ME).

Techniques: Immunofluorescence, Staining, Control, Translocation Assay, Electrophoretic Mobility Shift Assay, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot