Identification of RGNNV-induced ROS production and the effect of ROS on mitochondrial morphology and loss of ΔΨ in GF-1 cells.

<p>Phase-contrast and fluorescence micrographs showing ROS production (the Image-iT LIVE Green Reactive Oxygen Species Detection Kit) and mitochondrial morphology (stained by Mito tracker) were in the same cells. (A) RGNNV-infected GF-1 cells at 0 h (a–d), 24 h (e–h; ROS produced in cells, and 48 h (i–l). The elongated mitochondrial network at 0 h in A:d is indicated by arrow in A:p. ROS production at 0 h in A:m is indicated in open square in A:b; at 24 h pi in A:n and p is indicated open square in A:f and h; at 48 h pi in A:o and r is indicated in open square in A:j and l. Breakdown of mitochondrial fission (indicated by arrows) at 48 h pi in A:r is indicated open square in A:l. Scale bar = 10 µm. (B) RGNNV-infected GF-1 cells treated with NAC at 24 h (e–h) and 48 h pi (m–p), or not treated at 24 h (a–d) and 48 h pi (i–l) with RGNNV infection. Blockade of mitochondrial breakdown in RGNNV-infected GF-1 cells at 48 h pi in B:p is indicated open square in B:l, which were appeared some dot of mitochondria and indicated by arrow; without RGNNV-infected cells at 48 h pi in B:r is indicated open square in B:p, which have shown more longer mitochondria in length that indicated by arrow. Scale bar = 10 µm. (C) The effect of anti-oxidants NAC and DPI on ΔΨ in cells infected with RGNNV. The Δ<b>Ψ</b> (MMP loss) of RGNNV-infected GF-1 cells treated or not treated with NAC or DPI was determined at 0, 24, 48, and 72 h pi in triplicate. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.05.</p

Identification of RGNNV infection induces ROS production in GF-1 cells.

<p>(A) ROS production (indicated by arrows) at 0, 12, 24, 48, and 72 h pi by cells infected with RGNNV (MOI = 1). The negative control (0 h) (see e and i); 24 h negative control (see a and b); RGNNV-infected groups at 12, 24, 48, and 72 h pi (see f–m and j–n, respectively; Green fluorescent cells as the ROS production positive cells); positive control H₂O₂ (1 µM) at 24 h post-incubation (see c and d; Green fluorescent cells as the ROS production positive cells). Scale bar = 10 µm. (B) The percentage of ROS-producing cells was counted at 0, 12, 24, 48, and 72 h, and shows a significant increase over time. In this and all subsequent figures (unless otherwise noted) data are presented as the percentage of 200 cells at each time point determined in triplicate, with each point representing the mean of three independent experiments. The vertical bars indicate ± the standard error of the mean (SEM). All data were analyzed using either a paired or unpaired Student's <i>t</i>-test, as appropriate. Statistically significant was defined at <i>P</i><0.01. (C) The ratios of ROS-producing cells were counted by fluorescence microplate reader at 0, 12, 24, 48, and 72 h, and showed a significant increase at 24 h pi, but not at 48 h and 72 h pi because those times left few cells in wells. *<i>P</i><0.01 indicated a statistically significant difference between mean values of the groups. (D) Concentration of peroxide in medium of RGNNV-infected cells producing H₂O₂ ratio at 12, 24, 48, and 72 h pi, respectively. Peroxide concentration at each time point was determined in triplicate. *<i>P</i><0.05.</p

Identification of zebrafish catalase overexpression can reduce RGNNV-induced ROS-mediated cell death and viral titers in GF-1 cells.

<p>(A) Western blot analysis of zebrafish catalase-producing cell lines in GF-1 cells after selection with Zeocin (500 µg/ml). The stable clones are zfCatalase-1 (lane 2), zfCatalase-3 (lane 3), and vector control-4 (lane 1). HeLa cell lysate serves as a positive control (lane 4). Actin used as an internal loading control. (B) The number of ROS-producing cells after infection with RGNNV (MOI = 1) at 0, 48, and 72 h. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.01. (C) The viability of cells transfected with vector control-4 or zfcatalase-3 and infected with RGNNV was determined at 0, 48, and 72 h pi in triplicate by using a trypan blue dye exclusion assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025853#pone.0025853-Mullen1" target="_blank">[37]</a>. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.01. (D) Viral titers were assays in GF-1 cell line by using at 48 h and 72 h pi samples. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.05.</p

Influence of anti-oxidants NAC and DPI treatments on ROS production and cellular viability during RGNNV infection.

<p>(A) The GF-1 cells pre-treated with antioxidants either NAC or DPI for two hours, then infected with RGNNV (MOI = 1) for different time incubations. The number of ROS producing cells infected with RGNNV TN1 at 0, 24, 48, and 72 h pi was assayed with the Image-iT LIVE Green Reactive Oxygen Species Detection Kit. Data are the percentage of 200 cells at each time point, determined in triplicate, with each point representing the mean of three independent experiments; error bars represent the SEM. The data were analyzed using either a paired or unpaired Student's <i>t</i>-test. as appropriate. *<i>P</i><0.01. (B) The viability of GF-1 cells infected with RGNNV and treated with or without NAC or DPI at 0, 24, 48, and 72 h pi in triplicate by using a trypan blue dye exclusion assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025853#pone.0025853-Mullen1" target="_blank">[37]</a>. The data were analyzed using either a paired or unpaired Student's <i>t</i>-test. as appropriate. *<i>P</i><0.05.</p

Western blot analysis of RGNNV infection up-regulates anti-oxidant enzymes Cu/Zn SOD and catalase or transcriptional factor Nrf2 in GF-1 cells at middle replication stage.

<p>The GF-1 cells pre-treated with antioxidants either NAC (1 mM) or DPI (30 mM) for two hours, then infected with RGNNV (MOI = 1) for different time incubations at 0, 24, 48, and 72 h pi. Samples were electrophoresed on a SDS-polyacrylamine gel and electro-blotted to a NC membrane. The NC membrane was stained with mouse monoclonal IgG antibodies directed against Cu/Zn SOD (15-kDa; Cayman), Catalase (57-kDa; Rockland), and Nrf2 (74-kDa) (Rockland). The chemiluminescent signal was imaged on XAR-5 film (Kodak) using a 5-min exposure. Lanes 1–4, 30 ul of virus-infected GF-1 cells and corresponded to 0, 24 h, 48 h and 72 h pi, respectively. The actin internal is also shown.</p

Identification of anti-oxidants treatment can reduce apoptotic/necrotic death of cells infected with RGNNV.

<p>(A) Phase-contrast and fluorescent micrographs of annexin-V–stained, RGNNV-infected GF-1 cells without drug-treatment at 0 h (a and f), 24 h (b and g), 48 h (c and h), and 72 h (d and i) or with NAC-treatment at 24 h (e and j), 48 h (k and p), and 72 h (l and q) and DPA-treatment at 24 h (m and r), 48 h (n and s), and 72 h (o and t). Annexin-V–positive cells (necrotic cells) are indicated by arrows. Scale bar = 20 µm. (B) The number of annexin-V–positive cells after infection with RGNNV at 0, 24, 48, and 72 h. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.05. (C) Examples of flow cytometric profiles in 48 h pi. RGNNV-infected cell and plus anti-oxidants treatment cells PI staining fluorescence was measured from 10,000 cells. Numbers in second peak scales (PI⁺) show late apoptotic/secondary necrotic cell percentages respectively. Viable cell percentage (PI⁻) is shown in first peak.</p

Additional file 3: Figure S1. of Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection

Endogenous gene expressions of fatty acid synthesis genes were determined by real-time qPCR. The endogenous fatty acid synthesis genes, Fadsd2 (Fatty acid desaturase delta 2), Elovl2 (Elongase 2) and Elovl5 (Elongase 5) of zebrafish liver and muscle were analysis. (JPEG 301 kb