Human β-defensin-2 and IL-6 secretions by gingival epithelial cells were maintained over multiple cell passages following exposure to cigarette smoke.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 min or left untreated, followed by culture for 24 h, after which supernatants were collected and used to determine the levels of HBD2 or IL-6 by ELISA. Data are expressed as the means+SD (n = 5).</p

Whole cigarette smoke induced Toll-like receptor TLR2, 4, and 6 expression in normal human gingival epithelial cells.

<p>Gingival cells were exposed to whole cigarette smoke for 15 or 30 min or left untreated and subsequently cultured for various time periods prior to TLR gene expression analysis by qRT-PCR. TLR expression level at the cell membrane level was evaluated by flow cytometry. Representative histogram plot of each TLR expression: dark curve = non-exposed control cells; white curve = smoke-exposed cells. (n = 6); (Panel A), gene expression levels; (Panel B), protein expression. *, p<0.001.</p

The effect of whole cigarette smoke on TLR2, human β-defensin-2, and IL-6 expression by gingival epithelial cells was maintained over multiple cell passages.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 min or left untreated, followed by culture for 3 h, after which the cells were detached and used either to extract total RNA or to subculture for 4 days. This subculture was repeated twice every 3 days for a total culture period of 10 days. Extracted RNAs from the different conditions were analyzed by qRT-PCR. Data are expressed as the means+SD (n = 5); *, p<0.0001.</p

Whole cigarette smoke promoted human β-defensin-2 and -3 secretions through the ERK1/2 MAP kinase and NFκB signaling pathways.

<p>Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NF-κB inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 mn. 24 hours later, supernatants were collected and used to measure the β-defensin secretion levels by ELISA kits. Data are expressed the means+SD, (n = 4).</p

Human IL-1β and IL-6 gene and protein expression levels increased after normal human gingival epithelial cells were exposed to whole cigarette smoke.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 or 30 min or left untreated and then cultured for various time periods prior to gene expression analysis by qRT-PCR (5a and 5c). Data are expressed as the means+SD (n = 5); *, p<0.01. IL-1β and IL-6 levels were quantified by ELISA (5b and 5d) of the cells exposed to cigarette smoke and post-cultured for 24 h. Data are expressed the means+SD, (n = 6). **, p<0.001.</p

Human β-defensin-2 and -3 gene and protein expression levels increased when normal human gingival epithelial cells were exposed to cigarette smoke.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 or 30 min or left untreated and subsequently cultured for various time periods prior to gene expression analysis by qRT-PCR (3a and 3c). Data are expressed as the means+SD (n = 5); *, p<0.01. The β-defensin secretion levels were quantified by ELISA (3b and 3d) of the supernatant of the cells exposed to cigarette smoke and post-cultured for 24 h. Data are expressed the means+SD, (n = 6). **, p<0.0001.</p

Primer sequences used for the qRT-PCR.

<p>Primer sequences used for the qRT-PCR.</p

Whole cigarette smoke promoted human β-defensin-2 and -3 expression through the ERK1/2 MAP kinase and NFκB signaling pathways.

<p>Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and HBD gene expression was analyzed by qRT-PCR (n = 6). *, p<0.05; **, p<0.01 when comparing values obtained in the presence and absence of inhibitor. The results are expressed as the means+SD, (n = 5).</p

The dbSNP were used to recognize the protein encoded by PARP1 gene (PDB ID: 1uk0) and identified a single mutation residue position.

<p>The Z-score, which indicates overall model quality was -9.51 in (black color). The Z-score plot from the different sources (X-ray, and NMR) was distinguished by various colors (X-ray in pale blue and NMR in dark blue color).</p

The MD simulation showing truncated octahedron boundary explicit water solvated.

<p>The molecular dynamic simulation used in system calculation are, (a) water box surround the entire protein in middle. The visual inspection also allow to identify the side chain of the histidine residue involved in the hydrogen bonding with surrounding molecules and in that case the δ nitrogen of the histidine (HSB;1-4) was protonated residue.</p