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. 2017 Oct 6;13(10):e1006674. doi: 10.1371/journal.ppat.1006674

Fig 7. p97 and its ATPase activity are essential for EV71 replication, and it co-localizes with EV71 2C.

Fig 7

(A) RD cells were transfected with control siRNA or siRNA targeting Ubc6e or Herp. At 36 h post-transfection, cells were infected with EV71 (MOI = 10) for 12 h. Then real-time PCR was used to qualified viral RNA replication. Data are expressed as fold-change of the EV71 RNA level relative to cells transfected with siRNA control. The data were examined in at least two independent experiments; NS, non-significant, P≥0.05. (B) RD cells were transfected with control siRNA and siRNA targeting different ERAD components including EDEM1, OS9, SEL1L, Derl1, Derl2, Hrd1, gp78, RNF5, UBE2G2, VIMP, UBXD8, p97, Npl4, and Ufd1. At 36 h post-transfection, cells were mock infected (−) or infected (+) with EV71 (MOI = 10) for 12 h. Then, western blotting was carried out to detect the knockdown efficiency of different molecules, EV71 2C, and actin. (C) RD cells were pre-treated with increasing doses of DBeQ (0–10 μM) for 3 h and then mock-infected (−) or infected (+) with EV71 (MOI = 10) for 12 h. The cell lysates were then analyzed by western blotting with the indicated antibodies. Herp expression was used as an indicator of DBeQ efficiency, and actin was used as the loading control. (D) RD cells were transfected with plasmids encoding GFP or p97QQ-FLAG (dominant negative p97 mutant). At 36 h after transfection, the cells were mock-infected (−) or infected (+) with EV71 (MOI = 10) for 12 h, and the cell lysates were analyzed by western blotting with the indicated antibodies. (E) RD cells were mock-infected or infected with EV71 (MOI = 10) for 12 h, and immunostaining was then performed to detect the intracellular distribution of p97 and EV71 2C (p97, green; 2C, red; nuclei, blue). Insets show magnified views of the merged channels in the boxed region. (F) RD cells were mock infected (−) or infected (+) with EV71 (MOI = 10) for 12 h, and cell lysates were immunoprecipitated (IP) with p97 mouse monoclonal antibody or control mouse IgG. Cell lysates and precipitates were analyzed by western blotting with antibodies against p97 and EV71 2C. (G) 293T cells were cotransfected with empty vector (control) or plasmids encoding 2C-HA and p97-FLAG. At 36 h after transfection, cell lysates were immunoprecipitated with HA antibodies. The cell lysates and precipitates were then analyzed by western blotting with antibodies against p97-FLAG and 2C-HA.