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

Fig 7. Knockdown of ATF4 or NRF2 increased SG formation in WNV-infected cells.

Fig 7

(A and B) BHK and C57BL/6 and IFNAR-/- MEFs cells in wells of a 6-well plate were transfected with 83 pmol/well of ATF4- or Nrf2-specific siRNA or control siRNA for 24 h. The cells were then mock-infected or infected with WNV (MOI of 1). Whole cell lysates was harvested at 24 hpi and analyzed by immunoblotting using anti-ATF4 or anti-NS3 antibody. (C) BHK cells were transfected with control siRNA or specific siRNA and 24 h later infected with WNV (MOI of 1). Culture fluids collected at different times after infection were analyzed for virus infectivity by plaque assay. (D) BHK cells in wells of a 24-well plate were transfected with 17.5 pmol/well of ATF4- or Nrf2-specific siRNA or control siRNA. At 24 hpi, cells were infected with WNV (MOI of 3) for 24 h. Some of the replicate cultures were then incubated with Ars for 30 min, treated and untreated cells were fixed, permeablized and processed for IFA using anti-G3BP antibody (green) and anti-dsRNA antibody (white). WNV-infected, SG-positive cells are indicated by an asterisk. The images were deconvolved. Scale bars, 11 μm. (E) The average number of SG-positive, WNV-infected cells per 100 cells from 2 or 3 fields was determined for each of 3 replicate experiments. (F) BHK cells were transfected with 17.5 pmol/well of ATF4-specific or control siRNA. At 24 h after transfection, cells were infected with WNV (MOI of 3) and 24 h later incubated with ThiolTracker Violet in PBS for 30 min, washed and then fixed, permeabilized and processed for IFA. Anti-WNV antibody (white), anti-ATF4 (red), and ThiolTracker Violet intensity (blue). Scale bars, 11 μm. (G) The ThioTracker signal intensity in individual cells in the different categories in 2 fields from each of three biological replicate experiments (total 6) was quantified using Velocity software. The intensity data were plotted using GraphPad Prism 6. Symbols represent individual cell values. Long horizontal lines indicate mean values. Error bars indicate standard deviation (SD). **** P <0.0001.