Figure 4(A) The effects of prolonged exposure to gentamicin (300 µg/mL) or vancomycin (300 µg/mL) on the survival of phagocytosed S. aureus USA300 in RAW macrophages is shown. Depicted above the graph is a timeline detailing the treatment regime. The data are presented as the mean fold change in CFU/mL +/- standard deviation for 15 biological replicates of S. aureus, represented by each symbol, from five independent experiments. For each replicate the fold change was calculated by dividing the bacterial count at 10 h post-infection by the counts at 1.5 h post-infection. The dashed line indicates a fold change value equal to 1 (i.e. no change in bacterial burden). **p<0.01 and statistical significance was determined by a one-way ANOVA with a Dunnett's post-hoc test. (B) The ability to protect S. aureus USA300 from intracellular killing by gentamicin is shown. Here, experiments were performed as in (A) except during the initial gentamicin treatment (100 μg/mL for 1h) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK-III/Dynasore to inhibit fluid phase uptake. At 1.5 h post-infection cells were washed and treated with gentamicin (300 µg/mL) in the presence of either DMSO (vehicle) or PIK-III + Dynasore which were maintained throughout the experiment. The data are presented as the mean number of surviving bacteria in CFU/mL at 10 h post-infection +/- standard deviation. These data derive from a total of 15 biological replicates of S. aureus from at least three independent experiments. *** p<0.001 and statistical significance was determined by an unpaired two-tailed t-test with a Welch's correction. (C) The effect of PIK-III/Dynasore treatment on the ability of S. aureus USA300 to replicate within RAW macrophages is shown. After gentamicin treatment at 1.5 h, RAW 264.7 macrophages having phagocytosed S. aureus were lysed and plated to determine the number of gentamicin protected bacteria. Alternatively, infected samples treated with either DMSO as a vehicle control or with PIK-III/Dynasore which was added at 1.5h post-infection after gentamicin. At 10 h post-infection macrophages exposed to DMSO or inhibitors, that were maintained throughout the experiment, were lysed and plated to determine the bacterial burden. The data shown are the mean fold change in CFU/mL at 10 h where the data are normalized to the bacterial burden obtained at 1.5 h. Each symbol represents a biological replicate (n=8) of S. aureus and derive from three independent experiments. The data presented are the mean ± standard deviation. * indicates p<0.05 as determine by an unpaired two-tailed t-test with a Welch's correction. (D) The effect of PIK-III + Dynasore or DMSO on the growth of S. aureus USA300 in RPMI with 5% (v/v) FBS is shown. The data are the mean OD600nm +/- standard deviation of four biological replicates of S. aureus for each condition and are representative of three independent experiments. (E) Analysis of the effects of PIK-III/Dynasore treatment on S. aureus replication using a fluorescence based bacterial proliferation assay is shown. The micrographs depict S. aureus USA300 expressing GFP that were co-labelled with proliferation dye. The top row of micrographs depict vehicle treated macrophages where GFP-positive, yet proliferation dye-negative bacteria were present. The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive. The macrophage plasmalemma and extracellular bacteria are demarcated by TMR-WGA (in red). Images were acquired at 10 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm. (F) The normalized fraction of macrophages containing replicating bacteria is shown. The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition. Each symbol represents a biological replicate (n=6) of S. aureus and derive from three independent experiments. ****p<0.0001 and statistical significance was determined by an unpaired two-tailed t-test using a Welch's correction.