Anti vp1

The pre-cooled PBS was used to rinse the cells in 24-well plates (2 × 10⁴ cells/well) for 3 times and 4% paraformaldehyde was employed to fix the cells deposited in plates of 24-well. After that, cells were subjected to permeabilization with PBS containing 0.1% Triton X-100 for 10 min at 37 °C. Then, 3% BSA was added for blockading the nonspecific binding. Next, the cells were incubated with the anti-VP1 (1:1000 dilution; Millipore, USA), anti-cleaved-Caspase1 (1:100 dilution; Affinity, USA) and anti-NLRP3 antibody (1:100 dilution; Affinity, USA) overnight at 4 °C, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG and Daylight 594-conjugated donkey anti-rabbit IgG secondary antibodies (1:300 dilution; CST, USA) for 1 h. Finally, PBS was adopted to rinse the cells for 3 times and diamidino-2-phenylindole (DAPI; 1:1000 dilution; Beyotime, China) was taken to stain them at 37 °C for 5 min. With the help of a confocal fluorescence microscope (Leica, Germany), the images were obtained.

16HBE cells grown on sterile cover slips were treated with EV-A71 at 80% confluence for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with PBS containing 0.2% TritonX-100 for 10 min and blocked with 5% BSA for 30 min. After washed with PBS twice, cells were incubated overnight with anti-VP1 (1:1000 dilution; Millipore, USA), PRAKR1A (1:200 dilution; Affinity, USA), anti-JUNB (1:200 dilution; Affinity, USA) and anti-AHANK (1:200 dilution; Abcam, USA) antibodies followed by staining with FITC-conjugated donkey anti-mouse IgG and Daylight 594-conjugated donkey anti-rabbit IgG secondary antibody (1:100 dilution; Abbkine, China). Nuclei were stained with DAPI for 5 min, and the cells were washed 3 times. Cells were viewed under confocal fluorescence microscope (Leica, Germany).