Ev a71 cv a16 vp1

16HBE cells were added to 6-well plates and infected with EV-A71 or CV-A16. After treatment, cells were harvested, washed once with PBS and then fixed and permeabilized according to the BD Cytofix/Cytoperm™ Kit (BD Biosciences, USA) recommendations. Subsequently, cells were stained with EV-A71/CV-A16-VP1 (1:100, Millipore, USA) for 30 min at room temperature, and washed twice with PBS. Secondary antibody containing FITC marker was added, and the plate was incubated for 30 min in the dark at room temperature. After incubation, cells were resuspended in 0.5 ml of PBS and assayed by a FACScan flow cytometer (BD Biosciences, USA). Each sample contained 10,000 cells, and three independent replicates of each sample were tested. FlowJo 7.6 software was used for data analysis.

16HBE cells were seeded onto poly-L-lysine-coated coverslips (Solarbio, China) and treated as previously described. At the indicated time, the cells were washed in PBS, fixed with 4% PFA (Solarbio, China) and permeabilized with 1% Triton X-100 in PBS. The cells were blocked with 5% BSA at room temperature for 1 h and then incubated with the primary antibodies in blocking solution against EV-A71/CV-A16-VP1 (1:1,000, Millipore, USA), Nectin1 (1:100, Novusbio, USA), Claudin4 (1:200, Abcam, USA), E-cadherin (1:500, Abcam, USA) and ZO-1 (1:200, Abcam, USA) overnight at 4°C. Next, cells were washed with PBS three times and then incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Abcam, USA), Alexa Fluor 647-conjugated donkey anti-mouse IgG (Millipore, USA) or Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Biolegend, USA) for 1 h at room temperature. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:4,000, Beyotime, China). Slides were mounted with antifade reagent (Solarbio, China) and observed using a laser-scanning confocal microscope (Leica, Germany). The images were captured and processed using Adobe Photoshop 7.0 software.