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. 2018 Mar 23;8:91. doi: 10.3389/fcimb.2018.00091

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Copyright © 2018 Hayashi, Kubo, Izumida, Takahashi, Kido, Sato, Yamaya, Nishimura, Nakayama and Matsuyama.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Knockdown of endogenous EK in the A549 cells expressing sh-RIG-I reduced IAV proliferation. (A) sh-RIG-I-expressing A549 cells also expressing shRNAs targeting different sites in EK (sh-EK#1, sh-EK#2, or sh-cont.) were generated by infecting the cells with a recombinant lentivirus expressing an shRNA and a blasticidin-resistant gene. The drug-resistant cells were pooled and infected with IAV (MOI = 0, 0.001, 0.01, 0.1 from the left of ). At 48 h post-infection, the same cell lysate amounts were electrophoresed and western blotted with an anti-IAV HA antibody. The 65-kDa precursor IAV HA₀ and the cleaved 25-kDa C-terminal IAV HA₂ fragment are indicated by arrows. The HA₀ and HA₂ intensities and the HA₂/HA₀ ratio are shown below. (B) A549 cells (1 × 10⁵) in a 24-well plate were inoculated with IAV (MOI = 0.001). At 48 h post-infection, the viral spread was quantified as the release of infectious particles into the culture supernatants, as measured by a focus forming assay in MDCK cells. Data represent mean ± standard deviation of three experiments. ^*Significantly different at p < 0.05.

Inline graphic — Knockdown of endogenous EK in the A549 cells expressing sh-RIG-I reduced IAV proliferation. (A) sh-RIG-I-expressing A549 cells also expressing shRNAs targeting different sites in EK (sh-EK#1, sh-EK#2, or sh-cont.) were generated by infecting the cells with a recombinant lentivirus expressing an shRNA and a blasticidin-resistant gene. The drug-resistant cells were pooled and infected with IAV (MOI = 0, 0.001, 0.01, 0.1 from the left of ). At 48 h post-infection, the same cell lysate amounts were electrophoresed and western blotted with an anti-IAV HA antibody. The 65-kDa precursor IAV HA₀ and the cleaved 25-kDa C-terminal IAV HA₂ fragment are indicated by arrows. The HA₀ and HA₂ intensities and the HA₂/HA₀ ratio are shown below. (B) A549 cells (1 × 10⁵) in a 24-well plate were inoculated with IAV (MOI = 0.001). At 48 h post-infection, the viral spread was quantified as the release of infectious particles into the culture supernatants, as measured by a focus forming assay in MDCK cells. Data represent mean ± standard deviation of three experiments. ^*Significantly different at p < 0.05.