Briefly, RAW 264.7 cells grown on a 75 cm2 culture dish were seeded in 96 well plates at a density of 2×105 cells/well. Adhered cells were then incubated for 24 h with or without 1 μg mL−1 of E. coli lipopolysaccharide (LPS), in the absence or presence of 10 μM recombinant crystallin. The activated continuously with E. coli lipopolysaccharide (LPS; 1 μg mL−1) for 24 h as the positive control. Nitrite in culture supernatants was measured by adding 100 μL of Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthyl]-ethylenediamine dihydrochloride in 5% phosphoric GS-7340 clinical trial acid) to 100 μL samples of the medium for 10 min at room temperature. The optical density at 570 nm was measured using a Multiskan RC photometric

microplate reader (Labsystems, Helsinki, Finland). A NO standard curve was made with sodium nitrite. The detection limit of the assay was 0.5 μM. Immunofluorescence microscopy to observe the formation of punctated

crystallin foci was conducted essentially as described previously [31]. Briefly, cells were seeded into wells of a six-well culture plate containing a glass coverslip in each well. After treatment, cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized in 0.2% Triton X-100. After blockage with blocking serum for 1 h, samples were incubated with a rabbit polyclonal anti-crystallin antibody overnight at 4 °C, followed by Alexa-Fluor 594-conjugated goat anti-rabbit secondary antibody at room temperature for 1 h. To stain the nuclei, 4′,6′-diamidino-2-phenylindole Arachidonate 15-lipoxygenase dihydrochloride was added to the cells, which were then incubated for another 15 min. Each coverslip HDAC inhibitor was then removed from the plate, mounted on a glass slide, and observed with an Olympus

IX70 fluorescence microscope. To assess the effect of nodavirus-infected grouper on ROS production, the ROS-sensitive fluorescent probe DCFDA was used. Nodavirus-infected cells increased ROS production by 2.4-fold, comparing to noninfected cells, whereas ROS production was blocked by the antioxidant compound N-acetylcysteine (NAC; Fig. 1A). Because nodavirus infection leads to an inflammatory response and an increase in the basal oxidative stress, the next experiment determined the formation of dityrosine, serving as a marker of oxidatively modified proteins, in cells exposed to oxygen-free radicals. Dityrosine was prepared by incubation of tyrosine with horseradish peroxidase in the presence of H2O2. Dityrosine could be distinguished by the intense 420 nm fluorescence, measurable upon ultraviolet spectroscopy over a wavelength range of 254–365 nm, as shown in Supplementary Fig. 1A. Tyrosine and dityrosine were analyzed by reverse-phase HPLC with an ultraviolet detection wavelength of 280 nm. A typical chromatogram is shown in Supplementary Fig. 1B. Tyrosine and dityrosine eluted at 5.2 and 6.8 min, respectively.