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. 2017 Feb 27;13(2):e1006240. doi: 10.1371/journal.ppat.1006240

Fig 6. ATF4 and Nrf2 are activated by WNV infection.

Fig 6

(A) BHK cells were mock-infected or infected with WNV at an MOI of 1. Some replicate cultures were treated with Ars (0.5 mM) for 30 min at different times after infection and then whole cell lysates were collected in RIPA buffer and analyzed by Western blotting using anti-ATF4 and anti-WNV NS3 antibodies. (B) BHK cells were infected with WNV (MOI of 3) for 24 h. The cells were fixed permeabilized and processed for IFA. The cells were visualized with a 63X oil immersion objective on a widefield fluorescence microscope and the images were deconvolved. Anti-ATF4 antibody (green). Anti-WNV NS3 (red). The fluorescence intensity of nuclear ATF4 in mock-infected and WNV-infected BHK cells was quantified in all cells in a category in two fields obtained from each of three biological repeats (total of 6) using Velocity software. **** P <0.0001. Scale bars, 18 μm. (C) C57BL/6, eIF2α mutant AA, and PERK -/- MEFs were infected with WNV (MOI of 3) for 24 h. The cells were fixed, permeabilized and processed for IFA. Anti-ATF4 antibody (green). Anti-dsRNA antibody (red). Scale bars, 11 μm. (D) Whole cell lysates were harvested from WNV-infected (MOI of 1) BHK cells at different times after infection and used for immunoblotting with anti-Nrf2 antibody. Some replicate cultures were treated with Ars for 30 min at different times after infection. (E) BHK cells were infected with WNV (MOI of 3) for 24 h and the intracellular location of Nrf2 was analyzed by IFA. Images were deconvolved. Anti-p-Nrf2 antibody (green). Anti-dsRNA antibody (red). Nuclei were detected with Hoechst 33342 (blue). Scale bars, 11 μm. (F) Mock-infected BHK cells were treated with Ars (0.5 mM) for 30 min, fixed and processed for IFA. Cells were visualized with a widefield fluorescence microscope and the images were deconvolved. Top row: Anti-dsRNA antibody (red), anti-ATF4 antibody (green), Anti-TIAR (white). Bottom row: Anti-dsRNA antibody (red), anti-Nrf2 antibody (green) and anti-TIAR antibody (white). Scale bars, 18 μm. (G) BHK cells were mock-infected or infected with WNV (MOI of 1) and the whole cell lysates prepared at different times after infection were analyzed by immunoblotting using antibodies specific for the antioxidant enzymes GPx, GCLC and SOD. The asterisks indicate uninfected cells with SGs.