Figure 4
Construction and characterization of the EV71 3Cpro activity reporter cell line. (A) Immunofluorescence assay (IFA) of the control HEK293T cells and the EV71 3Cpro activity reporter cell line HEK293T-i-3CS-GLuc2 cells. The primary antibody is mouse anti-Flag antibody and the secondary antibody is FITC-labeled mouse anti-IgG antibody. Scale bar: 40 μm. (B) The cleavage of i-3CS-GLuc2 in HEK293T-i-3CS-GLuc2 cells by EV71 3Cpro or the catalytically inactive 3Cpro mutant H40D detected by Western blotting assays. The blots of Anti-HA were cropped and the full-length blots were displayed in Supplementary Information. (C) The dose effect of EV71 3Cpro on luciferase activity in HEK293T-i-3CS-GLuc2 cells. Cells were seeded in 12-well plates and transfected with various amounts of p3C-HA. The results are presented as the means ± SD of triplicate measurements (Student’s t test; **p < 0.01, ***p < 0.001, ****p < 0.0001). (D) The dose effect of Rupintrivir on luciferase activity in HEK293T-i-3CS-GLuc2 cells with and without 3Cpro expression. Cells were seeded in 12-well plates, transfected with and without 300 ng of p3C-HA and treated with Rupintrivir at various concentrations. The results are presented as the means ± SD of triplicate measurements (Student’s t test; ns, nonsignificant, ***p < 0.001, ****p < 0.0001). (E) The dose effect of the EV71 MOI value on luciferase activity in HEK293T-i-3CS-GLuc2 cells. The results are presented as the means ± SD of triplicate measurements (Student’s t test; **p < 0.01, ***p < 0.001, ****p < 0.0001).
