AP-3 is required for perinuclear inflammasome positioning and limits autophagy induction after <i>Salmonella</i> Typhimurium infection in DCs.

<p><b>(A-C).</b> WT and pearl (pe) BMDCs expressing ASC-GFP were infected with flagellin-expressing mCherry-STm (stimulates NLRC4). Cells were fixed 1 h after infection, labeled with DAPI and analyzed by fluorescence microscopy. <b>A.</b> Representative images showing ASC speck (green) relative to STm (red) and nucleus (blue) in four infected WT and pearl DCs each. <b>B.</b> Quantification of perinuclear (within a radius of one μm from the nucleus) and non-perinuclear ASC specks in 20 cells per cell type in each of four independent experiments. <b>C.</b> Quantification of ASC speck distance to the nucleus in 15 cells per cell type in each of three independent experiments. <b>(D</b>, <b>E)</b>. WT and pearl BMDCs were infected with STm, and endogenous LC3 (and actin as a control) was detected by immunoblotting fractionated cell lysates at the indicated time points. <b>D.</b> Representative blot with positions of molecular weight markers (MW) indicated at left. <b>E</b>. Quantification of LC3-II band intensities from three independent experiments, expressed as fold increase relative to unstimulated cells and normalized to LC3-I and β-actin levels. (<b>F</b>-<b>I).</b> WT and pearl BMDCs expressing ASC-GFP alone or with mCherry-LC3 were infected with STm and analyzed by live fluorescence imaging (for LC3) or fixed immunofluorescence microscopy (for p62) 1 h later. <b>F.</b> Representative images showing ASC speck (green) and either LC3 puncta (red, <i>left panels</i>) or endogenous p62 puncta (red) and nuclei (blue; <i>right panels</i>) in infected cells. Corresponding DIC images show nuclear position. <b>G, H.</b> Quantification of LC3 (<b>G</b>) or p62 (<b>H</b>) puncta within a radius of 0.5 μm from the ASC speck (representative image shown at right) in 15 cells per cell type in each of 3 independent experiments. <b>I</b>, Quantification of total p62 puncta normalized to cell area. Data represent mean ± SD. Scale bar: 10 μm.**p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s005" target="_blank">S5</a></b>and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s006" target="_blank">S6</a> Figs</b>.