Cytotoxicity of TBK1 inhibitor (TBK1/IKKε-IN-2) at different concentrations and the interaction effect between the virus and agonist/inhibitor.

(A) 104 RD cells were seeded to each well of the 96-well plate and cultured for 24 h. Then, cells were cultured with DMSO or TBK1 inhibitor (0–10 μM) for 24 h before performing CCK8 assays (Biosharp, Cat#: BS350B)) according to the supplier’s instructions. n = 6 wells/per group (B) The agonist/inhibitor are not directly viricidal. The virus (2.86 × 108 TCID50/mL) with or without agonist/inhibitor (100μM) was mixed and incubated for 1h at 37°C. We incubated the virus or the virus-agent mixture (MOI = 1) with monolayer RD cells for 1h at 4 °C (n = 4 wells/per group). After removing the culture medium, the cells were washed three times with cold PBS to remove the unbound viral particles. At 12 hpi, we freeze-thawed RD cells three times to assay the TCID50 of RD cell lysates and quantify the effect of the agonist/inhibitor on EV-A71 infectivity. Results were normalized by the infectivity of the original EV-A71. Statistical analysis was carried out using two-tailed unpaired Student’s t test. data represent the mean ± SD, ns, no significant. (TIF)