Study of cell migration and cytokine expression in pathogen infected oral keratinocytes.

<p><b>A.</b> Cell migration was assessed using transwell migration of keratinocytes after incubation with bacteria. The number of cells was counted by DAPI staining of nuclei using NIS element software under fluorescence microscope. Values were expressed as percent maximum against untreated control. Data are means ± SEM and shows one way ANOVA, with level of significance *p<0.05. <b>B.</b> Quantitation of integrin beta-3 at the protein level was determined by measuring fluorescence intensity of integrin beta-3 normalized to cell numbers. Values represent means ± SEM from three independent experiments. Significant between control and treatment *p<0.05 and between the two treatment groups +p<0.05 was determined by Ttest. <b>C–D.</b> Gene expression analysis of integrin beta-3 and -6 on day 1 and 3 post infection by real time RT PCR. The data is average from three independent experiments, δδCt gene/L32 value of each gene was calculated with normalization against expression of L32 control gene. Statistical significance represented by *p<0.05, and significant difference between expression on day 1 and 3 in a treatment group is represented by +p<0.05 after Tukey HSD test. <b>E–F</b>. mRNA levels of TNFα and IL-6 were measured by real time RT-PCR. Data shown are mean from three independent experiments. Statistical significance in differences of expression against control is represented by *p<0.05 after Ttest.</p

Delay in gap closure in pathogen infected scratch.

<p><b>A.</b> Scratch assay for assessing wound healing in periopathogen challenged primary gingival keratinocytes. The scratch width at the beginning of the scratch assay is represented by D0, wound healing on day 4 is represented in cells with no treatment (control), <i>P. gingivalis</i> and <i>F. nucleatum</i> challenge. Straight line represents the initial scratch boundary while curved line represents the leading edges of the healing wound. <b>B.</b> Graph shows scratch assay in oral primary keratinocytes where gap filling was measured based on the distance between the leading edges of the scratch. The data is average ± SEM from three independent experiments and shows ANOVA using Tukey HSD test. Significant difference between control and bacteria treatment is represented by *p<0.05. Significant difference between bacteria alone and bacteria along with Leupeptin treatment for a time point is represented by +p<0.05. <b>C.</b> CFSE based fluorescence of <i>P. gingivalis</i> co-localizing with actin was used to confirm presence of bacteria inside keratinocytes incubated without and with leupeptin. Bacteria inside keratinocyte is indicated by arrows. <b>D.</b> Graph represents the number of bacteria found within infected keratinocytes over period of 9 days thereby indicating the internalization of bacteria in medium without and with leupeptin. Values represent the average from three independent experiments.</p

Assessment of cell proliferation in bacteria infected oral keratinocytes.

<p><b>A.</b> Cell proliferation during wound healing was visualized using antibody against PCNA (b, e and h) and total number of cells in the field were marked by staining nuclei with DAPI (a, d and g). In primary gingival epithelial keratinocytes cell proliferation was assessed based on PCNA staining in regions R1, R2 and R3. The isotype control on bacteria challenged scratch assay for PCNA staining is represented by (k) with (j) showing DAPI staining. <b>B.</b> Graph representing cell proliferation at different regions of the scratch and at different time points. The total number of cells was counted based on DAPI staining of nuclei and cell proliferation was counted based on positive PCNA staining. D1 and D2 = day 1 and 2 in the graph. Average was calculated from three independent experiments ± SEM and shows one way ANOVA. Significant difference between control and <i>P. gingivalis</i> or <i>F. nucleatum</i> is represented by *p<0.05, scratch edge and other regions of a treatment group for the same time point is represented by +p<0.05. For a particular region significant between <i>P. gingivalis</i> and <i>F. nucleatum</i> is represented by ++p<0.05.</p

Analysis of expression of cell cycle genes in oral pathogen infected keratinocytes.

<p>Real time RT PCR of gene expression of Cyclin1 (A), cell division kinases 1, 2 and 4 (B, C, D) and cyclin dependent kinase inhibitor P18 (E) in bacteria challenged cells. Graph represents level of gene expression on day 1 and 3 post infection compared to day zero, i.e. prior to bacteria challenge. The data is average from three independent experiments. The δδCt gene/L32 value of each gene was calculated with normalization against expression of L32 control gene. Statistical significance represented by *p<0.05 and significant difference between expression on day 1 and 3 within a treatment group is represented by +p<0.05 after Tukey HSD test.</p

<i>P. gingivalis</i> up-regulates FOXO1 and FOXO3 mRNA levels.

<p>Primary human gingival epithelial cell cultures were incubated with or without <i>P. gingivalis</i> at MOI = 1∶50 for 20 hrs. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) FOXO1 prior to stimulation with bacteria. FOXO1 and FOXO3 mRNA levels were measured by real-time PCR. * Significantly different from control cells without bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05). + Significantly different between FOXO1 siRNA and FOXO3 siRNA (P<0.05).</p

<i>P. gingivalis</i> up-regulation of keratinocyte differentiation markers is FOXO1 or FOXO3 dependent.

<p>Primary human gingival epithelial cell cultures were incubated with or without <i>P. gingivalis</i> at MOI = 1∶50 for 20 hrs. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) FOXO1 prior to stimulation with bacteria. Real-time PCR was used to measure mRNA levels of keratin-1, keratin-10, involucrin and keratin-14. * Significantly different from control cells without bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05). + Significantly different between FOXO1 siRNA and FOXO3 siRNA (P<0.05).</p

<i>P. gingivalis</i> but not <i>F. nucleatum</i> or <i>S. gordonii</i> induces expression of differentiation markers.

<p>Primary human gingival epithelial cell cultures were incubated with or without exposure to an air-liquid-interface to induce differentiation and then challenged with <i>P. gingivalis</i> (Pg), S. <i>gordonii</i> (Sg), or <i>F. nucleatum</i> (Fn) at 2×10⁸/cm² for 24 hrs. mRNA levels of keratin-1 or keratin-10 were measured by real-time PCR. +Significant difference between undifferentiated and differentiated cells (P<0.05). * Significantly different from matched control (P<0.05).</p

<i>P. gingivalis</i> induces gingival epithelial cell apoptosis through FOXO1 or FOXO3.

<p>Primary human gingival epithelial cells were incubated with or without P. gingivalis at MOI = 1∶10 or 1∶50 for overnight. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) prior to incubation with bacteria. Apoptotic cells were assessed by the TUNEL assay. * Significantly different from control cells with bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05).</p

FOXO1 and FOXO3 regulate basal and <i>P. gingivalis</i> up-regulated mRNA levels of TLR2 and TLR4.

<p>Primary human gingival epithelial cell cultures were incubated with or without <i>P. gingivalis</i> at MOI = 1∶50 for 20 hrs. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) FOXO1 prior to stimulation with bacteria. Real-time PCR was used to measure mRNA levels of TLR-2 and TLR-4. * Significantly different from control cells without bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05). + Significantly different between FOXO1 siRNA and FOXO3 siRNA (P<0.05).</p