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Infection of TIGK epithelial cells with P. gingivalis expressing SerB diminishes IL8 promoter activity and phosphorylation of NF-κB p65.

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posted on 2013-04-18, 01:37 authored by Hiroki Takeuchi, Takanori Hirano, Sarah E. Whitmore, Ichijiro Morisaki, Atsuo Amano, Richard J. Lamont

(A) TIGKs were transiently transfected with the pIL-8 κB-Luc luciferase reporter plasmid for IL8 promoter activity, or pRL-CMV (CMV) in which Renilla luciferase is driven by the cytomegalovirus promoter, and infected with P. gingivalis WT or ΔserB mutant at a MOI of 10. At 16 h after infection, TIGKs were stimulated with TNF-α (10 ng/ml) for 3 h or left unstimulated and luciferase activity was measured and normalized to Renilla luciferase. Results are presented as fold relative to the activity of the non-stimulated control. Values are mean ± SD; n = 3; *, p<0.05. Data shown are representative of 2 biological replicates. (B) Immunoblots (IB) of lysates of TIGKs infected with P. gingivalis WT or ΔserB at a MOI of 10. At 16 h after infection, TIGKs were stimulated with TNF-α (10 ng/ml) or left unstimulated for the indicated time periods prior to cell lysis. Blots were probed with the antibodies indicated. Actin was used as a loading control. Result is representative of 2 biological replicates. (C) Densitometry of immunoblot in B) showing ratio of phospho-NF-κB p65 (S536) relative to total immunodetectable NF-κB p65. (D) Confocal microscopy showing expression of SerB by intracellular P. gingivalis. TIGKs were infected with P. gingivalis expressing FLAG-SerB at a MOI of 10 for 2 h, and stained with DAPI (cyan), fluorescein isothiocyanate (FITC)-phalloidin (green) or anti-FLAG (magenta). Bar = 5 µm. (E) Higher magnification of P. gingivalis indicated by arrow in D). Bar = 1 µm.

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