Additional file 6 of Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity

Additional file 6: Fig. S4. JEV infection induced the upregulation of IFI35 in hBMECs. hBMECs were infected with JEV P3 for the indicated time, and the expression of IFI35 at the mRNA level (A) and protein level (B) was measured by performing RT-qPCR and Western blotting, respectively. (C) HEK293T cells were transfected with an empty vector (vector) or IFI35 (Vector-IFI35) as indicated, and the expression of IFI35 and JEV-E at the protein level was measured by Western blotting. Data are represented as the mean values ± SEMs from three independent experiments. **p < 0.01; ****p < 0.0001. Fig. S5. JEV infection triggers the induction of ISGs in hBMECs, Related to Table 2. The hBMECs were infected with JEV P3 for the indicated time, and mRNA levels of IFN-λ2,3, IFITM1, ISG15, OAS1, OAS2, OASL, GBP4, CXCL10, USP18, IFI44, and CCL5 in these cells were quantified by RT-qPCR (A), and the protein levels of USP18, IFITM1, and ISG15 were measured by Western blotting (B). Fig. S6. JEV infection induced IRF activation in hBMECs. The hBMECs were infected with JEV P3 for the indicated time, and the expression of IRF7 and IRF1 at the mRNA level (A) and IRF7, IRF1, and IRF3/p-IRF3 at the protein level (B) were measured by performing RT-qPCR and Western blotting, respectively. (C) Cells were infected with JEV P3 for 36 h, and the protein levels of IRF7, IRF1, and IRF3/p-IRF3 were measured in shCTL, shTLR3, shRIG-I, and shMDA5 hBMECs, representing significantly upregulated and downregulated genes, respectively. Blue dots (middle) represent insignificantly expressed genes. Data are represented as the mean values ± SEMs from three independent experiments. ** p < 0.01; ****, p < 0.0001. Fig. S7. IFN-β/IFN-λ1 treatment induced IFIT mRNA expression in hBMECs. (A, C) hBMECs were treated with distinct concentrations of rhIFN-β/rhIFN-λ1 protein, and IFIT mRNA expression was measured. (B, D) Cells were treated with 10 ng/ml rhIFN-β (B) or 100 ng/ml rhIFN-λ1 protein (D) for different time points, and IFIT expression was measured by performing RT-qPCR. Data are represented as the mean values ± SEMs from three independent experiments. **p < 0.01; ****p < 0.0001.

补充材料6：图S4。日本脑炎病毒（Japanese Encephalitis Virus，JEV）感染可上调人类脑微血管内皮细胞（human brain microvascular endothelial cells，hBMECs）中IFI35的表达。将hBMECs以JEV P3毒株感染不同时长，分别通过逆转录实时定量聚合酶链反应（RT-qPCR）与蛋白质印迹法（Western blotting）检测IFI35在mRNA水平（A）与蛋白质水平（B）的表达情况。（C）将HEK293T细胞按实验分组转染空载体（vector）或IFI35过表达重组载体（Vector-IFI35），通过Western blotting检测IFI35与JEV包膜蛋白（JEV-E）的蛋白质表达水平。实验数据以三次独立实验的平均值±标准误（standard error of the mean，SEMs）表示。**p < 0.01；****p < 0.0001。**

图S5。JEV感染可诱导hBMECs中干扰素刺激基因（interferon-stimulated genes，ISGs）的表达，与表2相关。将hBMECs以JEV P3毒株感染不同时长，通过RT-qPCR定量检测细胞中IFN-λ2、IFN-λ3、IFITM1、ISG15、OAS1、OAS2、OASL、GBP4、CXCL10、USP18、IFI44及CCL5的mRNA水平（A），并通过Western blotting检测USP18、IFITM1与ISG15的蛋白质水平（B）。

图S6。JEV感染可激活hBMECs中的干扰素调节因子（interferon regulatory factor，IRF）通路。将hBMECs以JEV P3毒株感染不同时长，分别通过RT-qPCR与Western blotting检测IRF7与IRF1的mRNA水平（A），以及IRF7、IRF1与IRF3/p-IRF3的蛋白质水平（B）。（C）将细胞以JEV P3毒株感染36小时后，分别在阴性对照短发卡RNA转染组（shCTL）、靶向TLR3的短发卡RNA转染组（shTLR3）、靶向RIG-I的短发卡RNA转染组（shRIG-I）及靶向MDA5的短发卡RNA转染组（shMDA5）的hBMECs中检测IRF7、IRF1与IRF3/p-IRF3的蛋白质水平，上述基因分别为显著上调与显著下调基因。中间组蓝色圆点代表表达无显著差异的基因。实验数据以三次独立实验的平均值±SEMs表示。**p < 0.01；****p < 0.0001。**

图S7。干扰素-β（IFN-β）/干扰素-λ1（IFN-λ1）处理可诱导hBMECs中IFIT家族基因的mRNA表达。（A、C）将hBMECs用不同浓度的重组人IFN-β（rhIFN-β）/重组人IFN-λ1（rhIFN-λ1）蛋白处理，检测IFIT家族基因的mRNA表达。（B、D）将细胞用10 ng/ml的rhIFN-β（B）或100 ng/ml的rhIFN-λ1蛋白（D）处理不同时长，通过RT-qPCR检测IFIT基因的表达水平。实验数据以三次独立实验的平均值±SEMs表示。**p < 0.01；****p < 0.0001。**