Fig 1.

Lack of ATG5 decreases phagocytosis and intracellular killing of C. neoformans and C. albicans. (A) Two different shRNA sequences targeting murine ATG5 were transduced into J774.16 cells (clones 30 and 31), as was a scrambled shRNA control (C⁻). After selection with puromycin and cloning, J774.16 clones were evaluated by immunoblotting for ATG5 and β-actin expression. The proportion of ATG5 expression in comparison with the scrambled shRNA control was calculated by densitometry, and the two clones with the highest knockdown efficiency were selected for further experiments. (B) Antibody-opsonized C. neoformans phagocytosis after 2 h of coincubation using ATG5 shRNA-transfected J774.16 cells. The y axis represents the percentage of total macrophages with internalized C. neoformans cells. Bars represent the means of three independent experiments. (C) C. albicans phagocytosis after 30 min of coincubation with ATG5 shRNA J774.16 cells. The y axis represents the phagocytic index, calculated by dividing the number of internalized fungal cells by the total number of macrophages. Bars represent the means of five independent experiments. (D and E) C. neoformans killing assays after 24 h of incubation with ATG5 shRNA-transfected J774.16 cells. Monoclonal antibody and serum complement were used as opsonins in panels D and E, respectively. Bars represent the means and SEMs of 4 to 16 independent experiments. (F) C. albicans killing assay after 30 min of coincubation with ATG5 shRNA-transfected J774.16 cells. Bars represent the means and SEMs of 3 independent samples. (G and H) Secretion of IL-6 and IP-10 by shRNA-transfected cells. Each clone was incubated for 24 h with or without antibody-opsonized C. neoformans, and the supernatants were used for cytokine quantification. Bars represent the means and SEMs of 3 independent samples. *, P < 0.05 on the Bonferroni posttest that followed ANOVA analyses.