Figure 3.

DAPK1 Contributes to LC3-Dependent Aspergillus Autophagy

(A and B) GFP-LC3 punctae accumulation (A) (immunofluorescence) and percentage of GFP-LC3-positive cells (B) in RAW-GFP-LC3 cells after 1 hr stimulation with live A. fumigatus conidia + rIFN-γ in the presence of the DAPK inhibitor, Dapk1-specific siRNA (siDapk1), or negative control (Mock).

(C) LC3B-II/LC3B-I expression in RAW264.7 cells stimulated as above.

(D) Immunofluorescence imaging of RAW-GFP-LC3 or RAW264.7 cells pulsed with A. fumigatus conidia or inert beads in the presence of rIFN-γ.

(E–G) Purified phagosomes from RAW264.7 cells were pulsed with A. fumigatus conidia or inert beads for 1 hr in the presence of rIFN-γ or the indicated siRNAs to evaluate DAPK1 (E and G), Rubicon, Atg7, Beclin-1, and LC3B-II (F) expression by immunoblotting.

For immunofluorescence, nuclei were counterstained with DAPI. For immunoblotting, normalization was performed on mouse Gapdh (the corresponding ratio is indicated). Photographs were taken with a high-resolution microscope (Olympus BX51). Scale bar, 25 μm. BF, brightfield. Numbers refer to co-localization coefficients to quantify the overlap degree of DAPK1 and Beclin-1, Rubicon and Atg7. Data (mean values ± SD) represent pooled results or representative images (immunofluorescence and immunoblotting) from three experiments. ^∗∗∗p < 0.001, DAPK inhibitor or siDapk1 treated versus untreated RAW-GFP-LC3 cells. None, unpulsed and untreated cells. See also Figure S2.