FIGURE 5.

Src regulates phagolysosome acidification and Mtb targeting to the autophagosome. Localization of Mtb to the phagolysosome and autophagosome was monitored through confocal microscopy. A, PMA-treated cells were infected with PKH67-labeled Ra or Rv (green). Infected cells were maintained in the absence (−) or presence (+) of Src inhibitor PP2. At 48 and 90 h postinfection, cells were labeled with Lysotracker (marker for acidified lysosome; red) and the autophagy marker LC3 (blue). Cells were then fixed and examined under the laser-scanning confocal microscope at ×60 magnification. The effect of Src inhibition on the co-localization of Mtb with Lysotracker (Merge (Mtb:Lyso), yellow) or LC3 (Merge Mtb:LC3, cyan) is distinctly visible here. Rv-infected cells showed a marked increase in the yellow (with lysosome) and cyan (with autophagosome) colors, whereas Ra-infected cells did not show any significant effect upon Src inhibition. The co-localization coefficient for red-green (yellow) and blue-green (cyan) were calculated using the software Image-pro version 6.0. Data for the overlap coefficient (y axis) from more than 10 fields across two different experiments consisting of above 50 cells each are represented as bar plots in B (error bars represent S.E.).