Stimulation of interferon-stimulating genes (ISGs) in RAW264.7 cells.

<p>The time-dependent changes in mRNA expression after treatment with KIOM-C extract were confirmed by RT-PCR using the primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125357#pone.0125357.t002" target="_blank">Table 2</a>. All samples were normalized using GAPDH, and (A) equal amounts of PCR products were run on 1.5% ethidium bromide agarose gels and visualized using the GelDoc Imaging System. (B) The relative band intensities of IFN-β, IFN-α, TNF-α, IL-6, and IFN-related genes (Mx1, ISG15, and ISG56) were determined using the Image J analysis program and are shown as a bar graph. Error bars indicate the range of values obtained from three independent experiments.</p

Anti-viral activities of the KIOM-C extracts in RAW264.7 cells.

<p>Cells treated with media alone, 1 μg/ml KIOM-C extract, or 1000 U/ml recombinant mouse IFN-β 12 h prior to infection with (A) PR8-GFP, (B) VSVGFP or (C) NDV-GFP at an MOI of 0.1, 1.0 and 1.0, respectively. Images of GFP expression (left panel) were obtained 12 hpi (200 X magnification). Cell viabilities (middle panel) were determined by trypan blue exclusion at 24 hpi, presented as a percentage of the controls (cells without treatment). Viruses were titrated from the supernatant as PFU (right panel). In the case of NDV-GFP, the expression of NDV M mRNA over time in each treatment group was confirmed by specific PCR primers, which are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125357#pone.0125357.t001" target="_blank">Table 1</a>. All samples were normalized using GAPDH. Equal amounts of PCR products were run on 1.5% ethidium bromide agarose gels and visualized using the GelDoc Imaging System. The relative band intensity (RBI) of M mRNA expression from the same experiment is shown. RBI was determined (gene/GAPDH) using GelDoc Imaging System Band Quantification Software. Error bars indicate the range of values obtained from three independent experiments. Virus titers in the cell supernatant are expressed as means ± SD. Error bars indicate the range of values obtained from counting in triplicate during three independent experiments (*P<0.05 indicates a significant difference between groups compared by Student’s <i>t</i>-test).</p

Stimulation of an antiviral state by KIOM-C extract in RAW264.7 cells.

<p>Cells were treated with DMEM + 1% FBS alone, with 100 ng/ml LPS derived from <i>E</i>. <i>coli</i> or with 1 μg/ml KIOM-C extract and then incubated at 37°C with 5% CO2. Cells treated with DMEM + 1% FBS served as the negative controls. The supernatant from each group was harvested at 0, 12, and 24 hpt and clarified by centrifugation at 2500 X <i>g</i> for 10 min at 4°C. Ten-fold diluted supernatant was dispensed to the mouse. (A) The cytokines (TNF-α, IL-6, IL-12 or IFN-β) secreted in treated RAW264.7 cells were measured by cytokine ELISA using antibody-coated ELISA plates. The test was performed in triplicate. The data show representative means ± SD of each murine cytokine measured over time from three independent assays. The asterisk indicates a significant difference between groups (**<i>P</i><0.01; *<i>P</i><0.05). N.S., not significant. (B) Immunoblot analysis was performed on the cell lysates of RAW264.7 cells treated with or without KIOM-C to assess the expression of the phosphorylated and nonphosphorylated forms of IRF3, p65, STAT1, TBK1, p38, ERK and β-actin time dependently (h).</p

KIOM-C extract protects BALB/c mice against lethal infection with divergent influenza subtypes.

<p>BALB/c mice were treated orally with 200 μl/mouse of 100 μg/ml KIOM-C at 1, 3, and 5 days pre-; 1, 3, and 5 days post-; or both pre- and post-infection, along with (A) A/Aquatic bird/Korea/W81/2005(H5N2) (lower panel, log-rank test, <i>P</i> = 0.0016), (B) A/PR/8/34(H1N1) (lower panel, log-rank test, <i>P</i> = 0.0019), (C) A/Aquatic bird/Korea/W44/2005 (H7N3), (lower panel, log-rank test, <i>P</i> = 0.0006) or (D) A/Chicken/Korea/116/2004(H9N2), (lower panel, log-rank test, <i>P</i> = 0.0048) influenza subtypes. Mice treated with 0.85% normal saline (200 μl/mouse) prior to virus challenge were considered the negative controls (NC). Weight loss (upper panel) and percent survival (lower panel) were recorded until 13 dpi. (E) The virus titers in the lung tissues were measured by TCID50 at 3 and 5 dpi. (F) A representative image of the histopathological damage in eosin-stained lung tissue from mice treated with KIOM-C (pre-, post-, or pre- and post-infection) or 0.85% saline. Lungs of the KIOM-C-treated mice show clear alveoli without inflammatory cell infiltration, as opposed to the lungs of NC mice, which revealed features of severe pneumonitis. Tissues were observed under a light microscope at 200 X magnification. Bars denote means ± SD. Comparisons of groups were analyzed by Student’s t-tests and ANOVA. The asterisk indicates a significant difference between groups (*<i>P</i><0.05).</p