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Immunofluorescence microscopy. Raw264.7 cells grown on round coverslips in 12-well plates were allowed to phagocytose microbeads or infected with bacteria for the indicated times, fixed with 3% paraformaldehyde in PBS for 1 h at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and finally washed with PBS. Fixed cells were blocked with 3% bovine serum albumin in PBS for 1 h followed by staining with the anti-LAMP-2 (1:50 v/v), cathepsin D (1:50 v/v) JNJ26481585 for 1 h, and then incubated with Alexa488- or Alexa546-conjugated anti-IgG antibodies (1:1000 v/v) for 1 h.
Statistics. The unpaired two-sided Student’s t-test was used to assess the statistical significance of differences between the two groups.
Results and discussion
Rab7 transiently localizes on M.tb phagosome
Fig. 1.
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We investigated the dynamics of EGFP-Rab7 localization on M.tb phagosomes by live 4D confocal microscopy ( Fig. 1E and Movie S1). At 30 min postinfection, Rab7 was clearly present on the M.tb phagosome. The outline of the Rab7 signal surrounding the phagosome began to fade at 95 min and disappeared completely at 125 min. At 150 min postinfection, Rab7 was still absent from the phagosome. These results suggest orders Rab7 started to localize on the majority of M.tb phagosome immediately after infection, but viable M.tb subsequently caused the dissociation of Rab7 from the phagosome.