B3475

Cells were grown on glass slides in 24-well plates under standard conditions for 48 hr. Slides were then rinsed in PBS and fixed for 15 min in 4% PFA at 37°C. After washing with PBS, cells were permeabilized with PBS containing 0.2% Triton X-100% and 0.5% BSA for 5 min, and then blocked for 1 hr in PBS containing 0.5% BSA and 0.2% gelatine. Primary antibodies were added in blocking solution for 1 hr at room temperature in a humid chamber. The cells were rinsed in PBS three times before being incubated for 40 min in the dark with secondary antibodies and Phalloidin 568 (B3475 Thermo Scientific). After three washes with PBS, cells were stained for 5 min with PBS containing 0.1 µg/ml DAPI, followed by three washes with PBS. Glass slides were mounted with ProLong Gold Antifade Mountant (Thermo Scientific). The antibodies used for immunocytochemistry were: anti-CHORDC1 (HPA041040 Atlas Antibodies), anti-Tubulin (ab18251 abcam), anti-EGFR (MA5-13269 Thermo Scientific), anti-PDI (MA3-019 Thermo Scientific), Alexa Fluor 488 (A11034 Thermo Scientific), and Alexa Fluor 647 (A21236 Thermo Scientific). For all antibody stainings, at least three biological replicates were made.

Following removal of the cell media, cells were rinsed three times with cold 1X PBS, then fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS; pH 7.4) for 10 min at 37°C. PFA was removed and the cells were gently washed three times with 1X PBS. Cells were permeabilized by incubating in 0.1% Triton X-100 in 1X PBS at room temperature for 15 min. Following permeabilization, cells were again washed in 1X PBS three times then blocked in 2% BSA in 1X PBS at room temperature for 1 h. The NLRP3 primary antibody (ThermoFisher MA5-32255) was diluted per manufacturer’s protocol in 0.1% bovine serum albumin (BSA) in 1X PBS and incubated for 3 h at room temperature. Cells were washed three times with 1X PBS then incubated with secondary antibody (per manufacturer’s protocol; ThermoFisher A11008) and counterstained with phalloidin (ThermoFisher B3475) in 0.1% BSA in 1X PBS for 45 min. Cells were washed three times with 1X PBS and fluoromount media (ThermoFisher P36971) was added to each well to cover the cells. The Discover Echo Revolve microscope system was used to image the cells.