Figure 3. Endosomal vacuoles containing replication-deficient Francisella display features of autophagosomes. (A) Quantification of GFP-LC3 recruitment to LAMP1-positive vacuoles containing replication-deficient Francisella at 20 h and 24 h p.i. BMMs expressing GFP-LC3 were infected with either SchuS4 treated with chloramphenicol at 6 h p.i. or its isogenic ΔpurMCD or ΔdipA mutants, and processed for immunofluorescence labeling of bacteria, LAMP1-positive membranes and GFP. At least 30 LAMP1-positive bacteria per experiment were scored for LC3 recruitment in each condition. Data are means ± SD from three independent experiments. Asterisks indicate statistically significant differences (** p < 0.01, 1-way ANOVA, Tukey’s post-test). (B) Representative confocal (left panels) and transmission electron (right panels) micrographs of BMMs infected for 20 h with strains described in (A). Left panels, BMMs expressing GFP-LC3 were infected and processed for immunofluorescence labeling of bacteria (blue), LAMP1-positive membranes (red) and GFP (green). Magnified insets show single channel images of the boxed areas. Empty white arrows indicate bacteria enclosed within LAMP1-positive, LC3-negative vacuoles; solid white arrows indicate bacteria enclosed within LAMP1-positive and LC3-positive vacuoles. Right panels, BMMs were infected and processed for TEM as described in Materials and Methods. Insets show a magnification of the boxed areas. Black arrows indicate double membranes surrounding intracellular bacteria. Scale bars: 10 or 2 μm (confocal images) and 500 or 100 nm (TEM images).