Characteristics of the Study Subjects.

*<p>Mann-Whitney U test for continuous data, and Fisher's exact test for binary data.</p>**<p>Measured at the sampling sites.</p

Wnt5a was specifically up-regulated in THP-1 cells by <i>P. gingivalis</i> LPS.

<p>(A) HGF-1 and THP-1 cells were stimulated with <i>A. actinomycetemcomitans</i> sonicated extract, <i>P. gingivalis</i> sonicated extract, <i>P. gingivalis</i> LPS, and TNF-α for 4 hrs, and the expression of Wnt5a mRNA was detected by RT-PCR. PCR products were electrophoresed on a 1.5% agarose gel and visualized by UV illumination. GAPDH served as the internal control. (B) THP-1 cells were stimulated with 1 µg/ml of <i>P. gingivalis</i> LPS for 0.5, 2, 4, 12, or 24 hrs, and the expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are shown. (C) THP-1 cells were stimulated with 0.01–10 µg/ml of <i>P. gingivalis</i> LPS (black bars) or <i>E. coli</i> LPS (gray bars) for 4 hrs, and then the expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. (D) THP-1 cells were stimulated with <i>E. coli</i> 055:B5 LPS (middle and right upper panels) or <i>P. gingivalis</i> LPS (middle and right lower panels) for 30 min or 4 hrs, and then the expression of surface TLR2 and TLR4 protein was determined by flow cytometry. Left upper panel shows no-staining condition, and left lower panel shows un-stimulated condition with staining. (E, F, G, H) The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a mRNA are shown. (E) THP-1 cells were stimulated with 10⁴–10⁷ cells/ml of live <i>P. gingivalis</i> for 4 hrs. (F, G) Primary human gingival fibroblasts (HGF) and human monocytes were stimulated with 1 µg/ml of <i>P. gingivalis</i> LPS for 4 hrs. Monocytes were pretreated with the NF-κB inhibitor MG132 for 1 hr. Here we describe typical dates of three samples. (H) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after being transfected with TLR2 siRNA, TLR4 siRNA or control siRNA for 72 hrs. *p<0.05.</p

Induction of Wnt5a expression is partly JAK/STAT dependent.

<p>(A–D) The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. (A, B) THP-1 cells were pre-treated with AG490 (A) or fludarabine (B) for 1 hr and then stimulated with <i>P. gingivalis</i> LPS for 4 hrs. DMSO is a solvent control for both inhibitors. (C) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after transfection with STAT1 siRNA or control siRNA for 18 hrs. (D) THP-1 cells were pre-treated with STA21 for 1 hr and stimulated with <i>P. gingivalis</i> LPS for 4 hrs. *p<0.05.</p

<i>P. gingivalis</i> LPS-induced Wnt5a expression was inhibited by wedelolactone or dnIκBα.

<p>(A, B) THP-1 cells were pre-treated with the IKK inhibitor wedelolactone or the IKK inhibitor VII for 1 hr. The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. DMSO is a solvent control for both inhibitors. (C) THP-1 cells were transfected with a dominant-negative form of IκBα (dnIκBα) or the parent plasmid (pUSE) for 12 hrs. Fold induction of Wnt5a mRNA expression is shown. *p<0.05.</p

Induction of Wnt5a expression is NF-κB dependent.

<p>(A) THP-1 cells were stimulated with <i>E. coli</i> LPS or <i>P. gingivalis</i> LPS for 30 mins or 4 hrs. Whole cell extracts were prepared and analyzed by Western blot by using antibodies against IκBα. β-actin served as the protein loading control. (B) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after being transfected with NF-κB reporter plasmid for 12 hrs, and the transcription activity was assessed by luminometer. The activity is represented by the relative luciferase activity. (C) Nuclear extracts were prepared and EMSA was performed with γ³²P-labeled oligonucleotides representing the NF-κB consensus sequence as a probe. Anti-p65 antibody was used for supershift assays. The lower arrow shows the DNA-protein complex, and the upper arrow shows the supershifted band. *p<0.05.</p

The levels of Wnt5a mRNA were significantly up-regulated in chronic periodontitis tissues.

<p>Upper panel; Total RNA was extracted from periodontitis tissues, and the expression of Wnt5a mRNA was detected by RT-PCR. PCR products were electrophoresed on a 1.5% agarose gel and visualized by UV illumination. β-actin served as the internal control. Results are representative of five patients (right panel). Lower panel; The relative mRNA levels of Wnt5a. The horizontal line within each box represents the median expression level in each group.</p