SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Shi, Chong-Shan; Nabar, Neel R.; Huang, Ning-Na; Kehrl, John H.

Immunofluorescence THP-1 cells transiently transfected to express GFP-8b or GFP were seeded on glass-bottom collagen coated microwell dishes (MatTek), differentiated with PMA, and incubated overnight with LPS (50 ng/ml) and a caspase-1 inhibitor (Z-VAD-FMK, 10 μM). The following day, the cells were washed, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% bovine serum albumin for 15 min. For immunostaining, the cells were incubated for 4 h at room temperature or overnight at 4°C with primary antibodies diluted in 1% bovine serum albumin/ PBS. Fluorescent images were collected after secondary antibody incubation using a Leica TCS-SP5-X-WLL confocal microscope equipped with an argon and white light laser (Leica Microsystems) at either 63X or 100X oil immersion objective (NA 1.4). The samples were excited with 488-or 561-nm laser lines. Image analysis was performed using Imaris 8.0.0 (Bitplane AG) and Adobe Photoshop CS3 (Adobe Systems). HeLa cells transiently transfected to express NLRP3-Ds-Red plus or minus GFP-8b were washed 6 h after transfection and plated in glass-bottom microwell dishes overnight. Plated cells were processed and imaged as outlined above the following day. For DNA staining, the cells were incubated with 30 nM DAPI (Sigma) in PBS for 30 min and washed prior to imaging.