FIG 6.

Autophagy protein LC3 accumulates within CCVs in GNPTAB^−/− HeLa cells. (A and B) LC3 levels are elevated within CCVs in GNPTAB^−/− cells. Cells at 3 dpi were fixed with methanol and fluorescently stained for LC3 and CD63. Images were analyzed for LC3 intensity per CCV. (C to E) Bafilomycin treatment, but not induction of autophagy, accumulates LC3 and CD63 within CCVs of parental cells. Parental cells at 3 dpi were amino acid starved (−aa) or treated with bafilomycin A1 (BafA) for 4 h and then fixed with methanol and stained for LC3 and CD63. Images were analyzed for CD63 and LC3 intensity per CCV. Graphs represent the means ± SD of ≥150 cells from 3 independent experiments. Statistical significance was determined by Student's t test (****, P < 0.0001). Nuclei were stained with Hoechst and C. burnetii with NMII-specific antibody. Scale bar, 10 μm.