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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 RIG-I enhanced IAV proliferation in A549 cells. (A) A549 cells stably expressing the control shRNA (sh-cont.) or the shRNA confirmed as targeting RIG-I mRNA (sh-RIG-I) were generated by infection with an shRNA-expressing recombinant lentivirus and drug selection, as described in the section Materials and Methods. To verify the shRNA targeting RIG-I, the cell lysates were subjected to western blotting with an anti-RIG-I antibody. The 110-kDa RIG-I band is indicated with an arrow, and the band intensity is shown below. (B) A549 cells were infected with IAV (MOI = 0, 0.001, 0.01, 0.1 from the left of ) for 48 h, and the proliferation of IAV was analyzed with western blotting using an anti-HA antibody. The 65-kDa precursor IAV HA₀ is indicated with arrows. Intensities of HA₀ and HA₂, and the HA₂/HA₀ ratio were shown below. (C) The indicated 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 RIG-I enhanced IAV proliferation in A549 cells. (A) A549 cells stably expressing the control shRNA (sh-cont.) or the shRNA confirmed as targeting RIG-I mRNA (sh-RIG-I) were generated by infection with an shRNA-expressing recombinant lentivirus and drug selection, as described in the section Materials and Methods. To verify the shRNA targeting RIG-I, the cell lysates were subjected to western blotting with an anti-RIG-I antibody. The 110-kDa RIG-I band is indicated with an arrow, and the band intensity is shown below. (B) A549 cells were infected with IAV (MOI = 0, 0.001, 0.01, 0.1 from the left of ) for 48 h, and the proliferation of IAV was analyzed with western blotting using an anti-HA antibody. The 65-kDa precursor IAV HA₀ is indicated with arrows. Intensities of HA₀ and HA₂, and the HA₂/HA₀ ratio were shown below. (C) The indicated 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.