Figure 1.

Infection of macrophages with H. capsulatum induces LC3 conversion and recruitment to fungus-containing phagosome. (A) Macrophages from WT mice were stimulated with or without (0 min) H. capsulatum (MOI = 5) for the indicated time. Cell lysates were extracted and analyzed by Western blotting. (B) Macrophages from WT mice treated with or without cytochalasin D (CCD, 10 μg/ml) were stimulated with H. capsulatum at the indicated yeast-to-macrophage ratios for 60 min. Cell lysates were extracted and analyzed by Western blotting. (C) Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 60 min. Cells were fixed and stained for LC3B (green), F-actin (red), and nucleus compartment (blue). Cells were viewed under confocal microscope. Asterisks in the DIC/Nucleus field point to H. capsulatum yeasts. Box areas are shown at higher magnification in the bottom left corner of the corresponding image. The intensity of different fluorochromes along the white arrow in the merged image is shown as the histogram on the right. Data shown are representative of at least 3 independent experiments with similar results. (D) Macrophages from WT mice were stimulated with or without H. capsulatum (MOI = 2) for 30 min. Transmission electron microscopy (TEM) were used to analyze H. capsulatum-containing vacuoles in macrophages. Representative TEM micrographs are shown. Box area in the middle panel is shown as magnified image in the right panel. (E) Macrophages from WT mice were transfected with control siRNA or siRNA against Rubicon (50 nM) for 72 h. Cells were then stimulated with or without (0 min) H. capsulatum (MOI = 5) for 30 and 60 min. After stimulation, cell lysates were collected and assessed by Western blotting. Data shown in the lower panel are relative intensity of LC3-II normalized against the corresponding β-actin, mean ± SEM are shown (n = 3) (A,B,E).