Fig. 5.
FCVs display autophagic features, and their biogenesis requires autophagy. (A) LVS-infected BMMs were labeled with MDC (blue) before fixation at 24 h p.i. and immunofluorescence staining of Francisella (green) and LAMP-1 (red). (B) BMMs were transduced to express GFP-LC3 or GFP-LC3ΔC22, G120A (GFP-LC3ΔC22) and infected with LVS for 24 h p.i. before fixation and immunostaining of Francisella (blue), GFP (green), and LAMP-1 (red). Insets are single-channel fluorescence images of FCVs. Arrows indicate FCVs. (C) Quantitation of MDC accumulation and recruitment of GFP-LC3 or GFP-LC3ΔC22, G120A on LAMP-1-positive FCVs at 24 h p.i. For each condition, 100 FCVs were scored per experiment. (D) Effect of autophagy inhibition on FCV formation. (Left) LVS-infected BMMs were treated with 5 mM 3-MA at 14 h p.i., and FCV formation was scored at 24 h p.i. As a positive control for autophagy inhibition, uninfected, GFP-LC3-expressing BMMs were left untreated or pretreated with 3-MA for 1 h, then starved for 4 h to induce autophagosome formation. (Right) Autophagy was then scored as the percentage of cells containing GFP-LC3-positive vesicles. Asterisks indicate data significantly different from untreated controls (P < 0.05, two-tailed unpaired Student’s t test).