5 μM hemin and 3 μM menadione. TSB blood agar plates (BAP) were made with the addition of 5% sheep’s blood and 1.5% agarose. The bacteria were inoculated from BAP into 5 ml of TSBY and cultured anaerobically for 18 to 24 h at 37°C, then diluted in TSBY and grown to early log phase. Bacterial cells were harvested by low-speed centrifugation and resuspended in α-MEM (alpha minimum essential medium). Bacteria were then diluted in α-MEM to generate the appropriate MOI (multiplicity of infection) for addition to osteoblast cultures. Bacterial inoculation To identify the receptors utilized by

P. gingivalis during invasion of osteoblasts, P. gingivalis was inoculated into 1-week-old osteoblast cultures at a MOI of 150 for 1 h. To evaluate osteoblast cytoskeleton rearrangement upon P. gingivalis infection, P. gingivalis

was inoculated CP-690550 into 1-week-old osteoblast cultures at a MOI of 150 for 30 min, 3 h or 24 h. For signaling pathway and apoptosis assays, bacteria were inoculated at a MOI of 150 for 3 h in 1-week old osteoblast cultures (designated as day 1 on bacterial inoculation), then every other day up to day 21. For all inoculations, the osteoblasts were washed with PBS and then incubated with viable P. gingivalis at 37°C in 5% CO2/95% air for the time periods described above. Osteoblasts were washed with PBS again and cultured in fresh α-MEM until the next inoculation. Controls were subjected to the same media change and wash conditions 4-Aminobutyrate aminotransferase without the addition of bacteria. Western blotting Primary find more mouse calvarial osteoblasts were isolated and plated in 6-well plates in DMEM supplemented with 10% FBS and antibiotics. After 1 week, the medium was changed to α-MEM supplemented with 10% FBS, 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate to induce the differentiation of osteoblasts. The medium was changed every other day thereafter. On each medium change day, viable P. gingivalis

33277 was inoculated into the cultures at a MOI of 150 for 3 h, and this procedure was carried out for 3 weeks. Protein was extracted from the cultures at the end of each week with ice-cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitors (1 μg/ml leupeptin, 0.5 μg/ml pepstatin, 0.7 μg/ml aprotonin, 0.5 mM phenylmethylsulfonyl fluoride), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.004% sodium azide) by shaking at 4°C for 15 min. The homogenates were centrifuged at 10,000 × g for 20 min at 4°C. The supernatant protein concentration was determined by BCA assay. Proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10–20% gels and transferred to nitrocellulose membranes.