FIG 7.

Bacterial uptake in osteoblasts is highly dependent on the amount of Fn covering the cells. A549 and pHOB cells were treated with Fn siRNA or control siRNA for 2 days. (A) Representative immunofluorescence microscopy images of Fn expression on pHOB. Cells were stained for Fn. siRNA treated A549 cells (B) or siRNA treated pHOB (C) were infected with S. aureus or S. carnosus TM300(pFNBA4). One hour post infection, extracellular staphylococci were removed by lysostaphin treatment. Subsequently, the host cells were detached and the number of host cells was determined. Host cells were lysed and the number of viable intracellular bacteria was assessed by plate counting. Numbers of intracellular bacteria in control cells were set at 100%. Data are means ± SD from at least three independent experiments. *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, one-way ANOVA followed by Dunnett’s multiple-comparison test. (D) By imaging flow-cytometric analysis, the uptake of S. aureus 6850-GFP in control pHOB was compared to Fn siRNA-treated cells after 1 h of infection, a lysostaphin step, and fixation. Data represent the means ± SD from three independent experiments. ***, P ≤ 0.001, unpaired t test. (E) By spot counting of imaging flow cytometry data, the amount of bacteria per cell in control pHOB and Fn siRNA-treated cells was compared. Data represent the means ± SD from 3 independent experiments. ***, P ≤ 0.001, two-way ANOVA followed by Bonferroni posttests.