Figure 3.

GzmA is internalized in infected monocytes. (A) In flow cytometry studies comparing surface vs. intracellular staining, the majority of GzmA is inside the monocytes after treatment (n=8 in at least two independent experiments; means and SEM; Wilcoxon matched-pairs signed rank test). Similarly, GzmA is shown to be adjacent to the nucleus of treated monocytes as seen by confocal microscopy. (B) Representative confocal microscopy image showing GzmA in red and DAPI (nuclear stain) in blue. (C) Representative confocal microscopy images from three separate experiments showing GzmA in red, DAPI (nuclear stain) in blue, and BCG-GFP in green. Comparing 30 min and 2 h post-infection, GzmA-treated cells show lower bacterial burden. (D, E) Image analysis isolating BCG-GFP+ areas at 16 h post-infection: superimposition of areas where BCG is present and GzmA. GzmA and BCG are not found in the same cells as visually represented in images and measured in graphs by looking at integrated density signal of BCG and its overlap with GzmA (E) (mean, at least three independent experiments) (*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant).