Figure 2.

Inflammasome activation leads to partial co-localization of autophagosomes and inflammasomes. (a) Confocal microscopy of differentiated THP-1 GFP-LC3 cells transfected with Cy3 labeled 1.5 μg/ml poly(dA-dT) for 3h. The yellow dots (Merge panel) indicate co-localization between green (LC-3) and red (Cy3) signals. (b) Confocal microscopy of differentiated THP-1 GFP-LC3 cells transfected with Cy3-poly(dA-dT) imaged every 15 minutes starting 1h after transfection. Coalescence between the Cy3 and GFP signal is shown in 60 and 75 minute panels. (c) Confocal microscopy of differentiated THP-1 GFP-LC3 cells transfected with poly(dA-dT) or sham transfected, fixed, and immunostained for ASC (red). The insert to the right is a 5X zoom of the indicated areas. (d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA-dT) and immunostained for Lamp-1 and ASC. Below each image is a z-axis projection. (e) Confocal microscopy of differentiated THP-1 GFP-LC3 cells treated with LPS (500ng/ml) +ATP (3mM) for 2h, immunostained for ASC (red), and imaged. The 1^st and 2^nd panels are 3D volume renderings reconstructed from z-stack images (2^nd panel is rotated 90^O and zoomed 3x). The same images are shown as a surface segmentation model in the 3^rd and 4^th panels. Scale bars shown are 10 μm. (f) Electron microscopy of differentiated THP-1 cells stimulated with LPS + ATP or transfected with poly(dA-dT) for 2h and immunostained for ASC visualized with 15 nm gold conjugated protein A. ASC immunostaining is indicated with red arrows. Scale bars are 500 nm.