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. 2017 Apr 3;214(4):919–929. doi: 10.1084/jem.20161270

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© 2017 Fu et al.

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Figure 1. — ZMPSTE24 protects against viral infection. (A) ZMPSTE24-FLAG was transfected with HA-tagged STING, IFITM1, IFITM2, or IFITM3 into HEK293 cells. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody and blotted with the indicated antibodies. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IP, immunoprecipitation; WB, Western blot. (B) A549 cells were stimulated with 5 U IFNβ for the designated times. IFITM1, IFITM2, IFITM3, ZMPSTE24, and control GAPDH mRNA levels were examined by real-time PCR (three independent experiments). Mean ± SD; *, P < 0.05. (C) Empty vector, ZMPSTE24-FLAG, or untagged ZMPSTE24 were transfected into HEK293 cells. After 24 h, cells were infected with 0.1 MOI PR8-GLuc for 16 h. Cell viability was determined by CellTiter-Glo, which was used for normalization (three experiments). Mean ± SD; *, P < 0.05. (D) A549 cells transfected with ZMPSTE24-FLAG (48 h) and then infected with 1 MOI PR8 for the indicated times (two experiments; percentage of positive cells from five fields ± SD are denoted). Bars, 100 µm. (E) A549 cells transfected with ZMPSTE24-FLAG were infected with 0.001 MOI WSN IAV for 12 h. Virus titers were determined by plaque assay. Data are representative of three experiments. Mean ± SD; *, P < 0.05. (F) A549 cells transfected with ZMPSTE24-FLAG were infected with 1 MOI IAV-GLuc, VSV-Luc, Sindbis-Luc, MLV-Luc, VACV-Luc, cowpox-Luc, and AdV5-Luc for 16 h (consolidated data). Each group was analyzed three times. Mean ± SD; *, P < 0.05. Viability assays of infected cells indicate <20% differences among groups. (G) GFP-FLAG or ZMPSTE24-FLAG was transfected in T98-G cells for 24 h before infection with 0.01 MOI MR 766 Zika virus. After 72 h, Zika virus titers were determined by plaque assay (three experiments). Mean ± SD; *, P < 0.05. (H) Wild-type, ifitmDel^−/−, and reconstituted MEFs were infected with 1 MOI AdV, VACV-Luc, or cowpox-Luc for 16 h. Relative luciferase activities were examined (three experiments). Mean ± SD; *, P < 0.05.