Poly l lysine coated coverslips

HUVECs were seeded onto poly-L-lysine-coated coverslips (Solarbio, China). At the indicated time, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and permeabilized with 1% Triton X-100 in PBS for 15 min at room temperature. After blocking with 5% bovine serum albumin, the cells were incubated with the primary antibodies in blocking solution against EV71/CA16-VP1 (1:1000 dilution; Millipore, USA), Nectin1 (1:100 dilution), Claudin4 (1:200 dilution), VE-cadherin (1:500 dilution), and ZO-1 (1:200 dilution) overnight at 4 °C. Next, the cells were washed with PBS three times and then incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:1000 dilution; Abcam, USA) or Alexa Fluor 647-conjugated donkey anti-mouse IgG (1:1000 dilution; Millipore, USA) for 1 h at room temperature. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:4000 dilution; Beyotime, China). Following three washes in PBS, the coverslips were mounted on slides with an antifade reagent (Solarbio, China). Finally, images of the cells were captured with a laser-scanning confocal microscope (Leica, Germany) and processed using Adobe Photoshop 7.0 software.

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 CV-A10-VP1 (1:1,000, Genetex, China) overnight at 4 °C. Next, cells were washed with PBS three times and then incubated with Alexa Fluor 647-conjugated donkey anti-mouse IgG (Millipore, 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.

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.