Fig. 1.
PI3P persists on phagosomes containing dead but not live M. tuberculosis var. bovis BCG. (A) RAW 264.7 macrophages were transfected with P40PX-EGFP, allowed to phagocytose either live or dead (heat-inactivated) Texas red-labeled BCG, and analyzed by 4D confocal microscopy. Shown is quantification of PI3P positivity of phagosomes containing live or dead BCG (n = 45 live, n = 19 dead). (Insets) GFP fluorescence of the PI3P probe (grayscale) (Left) and merged images of GFP and red mycobacterial fluorescence (Right). **, P < 0.01. (B) Temporal quantification of phagosome fluorescence intensity relative to fluorescence of the cytosol. Rφ/c, ratio between phagosome fluorescence intensity and cytosol fluorescence intensity. Shown are Rφ/c obtained by 4D microscopy and live imaging of three different phagosomes harboring dead BCG (filled squares) and three different phagosomes in cells infected with live BCG (open triangles). (C) Quantification of fluorescence levels over time, expressed in relative fluorescence units (RFU; subtracted for RFU of the cytosol) of a phagosome with dead (filled squares) or live (open triangles) BCG. (D) Confocal immunofluorescence images of fixed specimens containing macrophages infected with live or dead BCG and immuno-stained for CD63. (E) Quantification of CD63 staining of phagosomes.