Figure 3

Infection with S. gordonii antagonizes the effect of P. gingivalis on the cell cycle. HIGK (uninfected in A) were simultaneously grown in the presence of BrdU and infected with P. gingivalis labeled with CellTracker Green BIODIPY shown in blue (B), CellTracker Blue CMAC labeled S. gordonii shown in pink (C), or a combination of P. gingivalis and S. gordonii for 4 h (D). HIGK cells were treated with antibiotic to kill extracellular bacteria and were cultured for an additional 20 h prior to harvesting for FACS analysis. The cell cycle positions and active DNA synthetic activities of cells were determined by analyzing the correlated expression of total DNA (7-AAD) and incorporated BrdU levels. Uninfected control HIGK were included for comparison of baseline cycling patterns. Data represent duplicate experiments. Region gate 1, HIGK were apoptotic (defined as sub G₀/G₁); region 2, G₀/G₁; region 3, S phase; region 4, G₂+M. Panel E and F demonstrate the gated HIGK populations used throughout all the analyses for CellTracker Green labeled P. gingivalis and CellTracker Blue CMAC labeled S. gordonii, respectively. Panel G depicts quantitative analysis of HIGK cell cycling in response to mono- and mixed infections. Results are the mean of three experiments. Statistical analysis was conducted using an ANOVA with Bonferroni's Multiple Comparison Test. * Pg vs. Sg sub G₀/G₁, Pg vs. Sg G₀/G₁, Pg vs. Sg G₂+M, Pg vs. Sg S phase, p value < 0.05. # Pg vs. Sg+Pg sub G₀/G₁, G₀/G₁, G₂+M, S phase, p values < 0.001. Error bars represent the mean ± SD.