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. 2009 Nov 20;4(11):e7919. doi: 10.1371/journal.pone.0007919

Figure 8. Akt promotes the colocalization of FCP with lysosomes.

Figure 8

BMM from wild type (A) and Myr Akt (B) mice were pre-loaded with LysoTracker red for 30 min and subsequently infected with F. tularensis subsp. novicida at an MOI of 100 for 30 min. The extracellular bacteria were killed and removed by gentamicin treatment followed by washing. The infected cells were incubated for 2 h and then fixed. The preparation was incubated with mouse anti-F tularensis subsp. novicida monoclonal antibody followed by the addition of Alexa Fluor 488 goat anti-mouse IgG. The macrophage nuclei were stained with DAPI for 5 min at room temperature. The FCP (green)-lysosome (red) colocalization was examined using a LSM 510 confocal microscope (magnification ×26000). (C) The number of FCP-lysosome co-localization events was scored. Positive only when FCP fused completely with lysosomes (orange). The graph shows mean and SD of three independent experiments. 100 cells were scored in each experiment. * indicates p value <0.05. (D) In a parallel experiment the phagocytic index (uptake of bacteria) was analyzed by immunofluorescence microscopy. BMM were infected with F. tularensis subsp. novicida at a MOI of 100. After 30 min of infection, cells were fixed and immunostained with F. tularensis subsp. novicida LPS antibody followed by Alexa Fluor 488-conjugated secondary antibody. At least 100 cells per sample were examined and three separate sets of infections were analyzed. The graph shows the mean and SD of three independent experiments. * indicates a p value <0.05.