Osteoblast nuclei were labeled with DAPI (Molecular Probes). The confocal images were captured with an Olympus FV1000 Laser Confocal microscope using Olympus
Fluoview software (Olympus America Inc. Center Valley, PA). The potential binding between osteoblast integrin α5β1 and P. gingivalis fimbriae was indicated by the yellow fluorescence where red (α5β1) and green (fimbriae) fluorescence co-localized. To determine whether α5β1-fimbriae binding and/or new host protein synthesis were essential for P. gingivalis invasion of osteoblasts, four experimental groups were set up: 1) control, osteoblasts without P. gingivalis inoculation; 2) osteoblasts inoculated with P. gingivalis; 3) osteoblasts treated with a 1:100 dilution of rat anti-mouse integrin α5β1 monoclonal antibody (Millipore) LDE225 in vitro PS-341 research buy for 1 h at RT prior to bacterial inoculation; 4) osteoblasts pretreated with the protein synthesis inhibitor, cycloheximide (50 μg/ml), 1 h prior to bacterial inoculation. For groups 2, 3 and 4, osteoblasts were inoculated with P. gingivalis at a MOI of 150 for 30 min, 1 h and 3 h. Thereafter, the cultures were washed, fixed, permeabilized and blocked
as described above. The cells were incubated with rabbit anti-P. gingivalis polyclonal antibody (1:4000) for 1 hr at RT, followed by washing and incubation with Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:200; Molecular Probes) for 1 h at RT. Osteoblast actin and nuclei were labeled with rhodamine phalloidin (Molecular Probes) and DAPI, respectively. The internalization of P. gingivalis into osteoblasts was determined by the localization of the bacteria within the cytoplasmic boundary of osteoblasts, as well as the close proximity of the bacteria to osteoblast nuclei. The number of osteoblasts with bacterial invasion was counted manually and expressed as the percentage of
the total number PRKD3 of osteoblasts counted. To determine whether actin rearrangement is required for P. gingivalis invasion, osteoblasts were inoculated with P. gingivalis at a MOI of 150 for 30 min, 3 h and 24 h with or without the addition of the actin-disrupting agent, cytochalasin D (2.5 μg/ml), for the entire infection period. Uninfected osteoblasts were used as controls. The staining process and confocal image acquisition were performed as described above. The number of osteoblasts with bacterial invasion was counted manually and expressed as the percentage of the total number of osteoblasts counted. TUNEL staining P. gingivalis-infected osteoblast cultures were fixed with 4% PFA in PBS. The TUNEL procedure was performed with the TACS TBL kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Nuclease treatment or exclusion of TdT enzyme was used as the positive or negative control, respectively. Light microscopic examination revealed apoptotic cells as having condensed, blue-stained nuclei.
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