Figure 3: Defective autophagosome formation in Irf8^−/− MФs.

(a) LC3 vesicles were visualized in WT and Irf8^−/− MФs expressing mCherry-EGFP-LC3 vector, without (UT) or with IFNγ/TLR stimulation for 8 h. Bafilomycin A1 (BA1; 200 nM) was added for the final 2 h. Cells were counterstained for DNA (blue). Scale bar, 20 μm. Below: the number of cells with more than five mCherry-positive vesicles was counted by microscopic inspection of more than 200 cells. The values represent the percentage of cells with fluorescent vesicles. **P≤0.01 (Student’s t-test). See Supplementary Fig. 3a for endogenous staining of LC3. (b) WT and Irf8^−/− MФs treated with IFNγ overnight followed by TLR ligands for 8 h was inspected by transmission electron microscopy. The bracketed region in the left panel was enlarged in the right panel. Arrows indicate autophagic vacuoles. Scale bar, 0.5 μm. (c) Reduced LC3I to LC3II conversion in Irf8^−/− MФs. WT and Irf8^−/− MФs were treated with IFNγ/TLR as above with BA1 (200 nM) treatment for the final 2 h. Immunoblot analysis was performed with 10 μg of extracts with β-Tubulin as a control. Right panel: the amounts of LC3II in three independent samples were quantified using the ImageJ software. *P≤0.05 and **P≤0.01 (Student’s t-test). See Supplementary Fig. 3b for LC3 amount in the absence of BA1. (d) Membrane-bound LC3 in WT and Irf8^−/− MΦs treated with IFNγ/TLR was detected by flow cytometry. BA1 (200 nM) was added for the final 2 h. The histogram is a representative of three independent experiments. See Supplementary Fig. 3c for LC3 amount in the absence of BA1. (e) Immunoblot detection of the ATG5–ATG12 conjugate. WT and Irf8^−/− MФs were treated as above and immunoblot detection of the ATG5–ATG12 conjugate proteins was performed. Ten micrograms of the extracts were tested with antibody against ATG5 or β-Tubulin.