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: Cell reports

Article Title: Bladder epithelial cell phosphate transporter inhibition protects mice against uropathogenic Escherichia coli infection.

doi: 10.1016/j.celrep.2022.110698

Figure Lengend Snippet: Figure 5. UPEC infection triggers upregulation of PIT1 via NF-kB (A) Fold change in the mRNA levels of PIT1 in CFT073-infected 5637 cells pretreated with BAY11-7085 (left) or 5637 cells with silenced NF-kB p65 (right) (n = 3). (B) Phosphate concentration in BCVs purified from 5637 cells pretreated with BAY11-7085 (left) or 5637 cells with silenced NF-kB p65 (right) (n = 3). (C and D) Fold change in the mRNA levels of UPEC pitB (C) or pldA (D) in 5637 cells pretreated with BAY11-7085 (left) or 5637 cells with silenced NF-kB p65 (right) (n = 3). (E and F) Colocalization of CFT073 expressing mCherry (red) with Rab27b (E) or galectin-3 (F) (green) in infected 5637 cells pretreated with BAY11-7085 at 1 h p.i. Scale bar, 5 mm. The bar graph shows the percentage of intracellular bacteria that are colocalized with Rab27b (E) or galectin-3 (F) relative to total intracellular bacteria (n = 3). Data are represented as mean ± SEM. Significance is indicated as the p value.

Article Snippet: For western blot and immunofluorescence analyses, the following antibodies were used: rabbit anti-Rab27b (IBL, 18973), mouse anti-Rab27b (Abcam, ab76779), mouse anti-galectin 3 (Abcam, ab2785), rabbit anti-SLC20A1 (Abcam, ab237527), rabbit antiNF-kB p65 (CST, 4764), rabbit anti-GAPDH (CST, 2118), rabbit anti-groEL (Abcam, ab90522), goat anti-rabbit IgG Dylight594 (EarthOx, E032420), goat anti-mouse IgG FITC (Abcam, ab6785), goat anti-rabbit IgG Alexa Fluor 680 (Abcam, ab186696), and goat anti-rabbit IgG (HRP, Sparkjade, EF0002), goat anti-mouse IgG (HRP, ab6789).

Techniques: Infection, Concentration Assay, Expressing, Bacteria

Journal: Turkish Neurosurgery

Article Title: Effects of glycogen synthase kinase inhibitor on glioblastoma multiforme cell line via apoptosis and cell signaling pathways

doi: 10.5137/1019-5149.jtn.23987-18.2

Figure Lengend Snippet: Figure 4: Fluorescence microscope image of the change in; (A–B) the NF-κB p105/p50 protein and, (C–D) the EGFR protein in cell lines (A–C: SVGp12, B–D: U-87 MG) treated with GSK-3 inhibitor IX in 2.05-μM doses for 48 hours (scale bar: 100 μm).

Article Snippet: The antibodies used were antiNF-kB p105/ p50 (1:50 diluted; AM06029FC-N, OriGene Technologies, Rockville, MD), antiEGFR (1:50 diluted; TA303484, OriGene Technologies) and FITC Conjugated Goat Anti-Rabbit IgG (1:100 diluted; TA130022, OriGene Technologies).

Techniques: Fluorescence, Microscopy