Cell counting kit cck 8

(1) Cell Counting Kit (CCK‐8) (Biosharp) was adopted to detect cell proliferate ability. Cells (1000 cells in the Figure 2 and 5000 cells in the Figure 7) were seeded in 96‐well plates, and after treatment for 48 h, the medium was replaced with new culture medium supplementing with 10% CCK‐8. The cells were then incubated at incubator for 2 h. Then, the optical density value at 450 nm wavelength was measured on the microplate reader to assess the degree of yellow color in the medium.
(2) The EdU assay was detected by EdU proliferation kit (Ribo‐Bio). Briefly, after treatment for 48 h, cells were incubated with 50 µM EdU for suitable time. Then fixation, permeabilization, and EdU staining were undertaken following manufacturer's instructions. Subsequently, cell nuclei were stained with Hoechst 33342. Finally, the results were photographed by a Leica microscope and the proportion of EdU‐positive cells was examined.

Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China). The Cell Counting Kit (CCK-8) was obtained from Biosharp (Anhui, China). Pyridone 6 (JAK inhibitor I) (A13457) was purchased from Adooq Biosciences (Irvine, CA, USA). Antibodies used for Western blotting, anti-phospho-STAT1 (S727), anti-STAT1, and anti-IFI35 antibodies (all rabbit polyclonal antibodies), were obtained from Abmart (Shanghai, China). Anti-IFIT2, anti-TLR3, anti-IRF7, anti-IRF3, anti-USP18, and anti-ISG15 antibodies (all rabbit) were purchased from ABclonal (Wuhan, Hubei, China). Anti-JAK1, anti-JAK2, anti-phospho-JAK1, and anti-phospho-JAK2 antibodies (all rabbit) were purchased from Abcam (Cambridge, MA, USA). Anti-MDA5 (IFIH1) and anti phospho-IRF3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-RIG-I (DDX58), anti-IFIT1, anti-IFIT3, anti-IFIT5, anti-IRF1, anti-IFITM1, and anti-β-actin antibodies were purchased from Proteintech (Chicago, IL, USA), and the antibodies used are listed in Additional file 7: Table S7. The monoclonal antibody against the JEV envelope (E) was kindly provided by Shengbo Cao (Huazhong Agricultural University, Wuhan, China). For immunofluorescence, rabbit anti-FLAG antibody was purchased from Proteintech.