Fig 2.

p62 is important for autophagic elimination of tubercle bacilli but does not function in the maturation of mycobacterial phagosomes. (A-D), Autophagy enhances p62 colocalization with mycobacterial phagosomes. RAW264.7 cells were infected with Alexa-568 labeled BCG and chased for 1 h in complete media. Cells were then subjected to autophagic induction for 4 h by starvation (A and B) or 24 h by IFN-γ treatment (C and D). Cells were fixed and stained for p62. (E - H) p62 does not play a role in mycobacterial phagosome maturation. RAW264.7 cells were transfected with siRNAs against p62 or scramble control. At 48 h after transfection, cells were infected with Alexa-488 labeled mycobacteria and chased for 1 h in complete media. Cells were subjected to autophagic induction by starvation for 4 h and stained with LysoTracker Red (E and F) or cathepsin D (G and H) to determine the acidification and acquisition of a lysosomal protease by mycobacterial phagosomes. Data, means ± SEM from at least three independent experiments. At least 50 phagosomes per category per independent experiment, were quantified using % BCG-marker colocalization; **, p < 0.01, *, p < 0.05, ^†, p ≥ 0.05, all relative to starvation control. For Pearson's colocalization coefficient (defined in Experimental Procedures) exact p values are shown and number of phagosomes analysed in each category indicated. See also Figs. S2 and S3, and Movies S1 and S2.