2.5. Luciferase Reporter Assay and ELISA

HEK293 cells seeded on 24-well plates (7 × 10⁴ cells per well) were transfected with a reporter plasmid (e.g., IFN-β-Luc or ISRE-Luc) with an internal control plasmid (pRL-TK) and the indicated plasmids by lipofectamine 2000. An empty vector (pcDNA6.0/Myc-His) was used to equalize the total amount of plasmids. After 24 h, the cells were infected with EV-A71 or treated as indicated stimulants. Heat-inactivated EV-A71 was derived from the incubation of live EV-A71 at 65 °C for 30 min. For an influenza virus infection, the cells were washed with PBS, and then infected with IAV (WSN) at the indicated titers in DMEM containing 2% bovine albumin. After indicated treatments, the activities of firefly and Renilla luciferases in lysates were measured using the Dual-Luciferase assay kit (Promega) according to the manufacturer’s instructions. The firefly luciferase activity was normalized by the Renilla luciferase activity, and the fold induction of each sample was calculated relatively to a control sample. The supernatants from treated cells were collected and analyzed for IFN-β and IFN-β inducible protein 10 (IP-10), and regulated, on activation, for normal T-cell expressed and secreted (RANTES) production by ELISA. The ELISA kits were used in the study, including human IFN-β (PBL), human IP-10 (R&D Systems), and human RANTES (R&D Systems). Values represent the mean ± standard error of the mean (SE) of the duplicated samples. Data are representative of two or three experiments.