24 HR - DMSO (10 fps)_A3_2

24 HR treatment of 0.5% DMSO control on neuroblastoma cells

24 HR - 50 uM MB

24 HR treatment of 50 uM MB on neuroblastoma cells

Mitochondrial metabolites from AE that were found to discriminate male from female mitochondria using false discovery rate analysis (q = 0.1).

<p>Metabolites with greater ion intensity in male mitochondria include (A) leucine/isoleucine, (B) glutamate and (C) methionine. Metabolites with greater ion intensity in female mitochondria include (D) adenosine, (E) amino octadecanoic acid and (F) unknown metabolite (<i>m/z</i> 537.789). Data was analyzed using one-way ANOVA and Tukey's post hoc test (* p<0.05, ** p<0.01, *** p<0.001).</p

A breakdown of the animals used for mitochondrial isolation and metabolic profiling studies.

<p>A total of 40 animals were used, 20 wild type and 20 thiroredoxin-2 transgenic (Trx2TG). These groups were further broken down into subgroups of 5 based on age and sex.</p

Mitochondrial metabolites from C18 that were found to discriminate male from female mitochondria using false discovery rate analysis (q = 0.1).

<p>Metabolites with greater ion intensity in male mitochondria include (A) leucine/isoleucine, (B) glutamate and (C) methionine. Metabolites with greater ion intensity in female mitochondria include (D) adenosine, (E) sphinganine and (F) unknown metabolite (<i>m/z</i> 442.759). Data was analyzed using one-way ANOVA and Tukey's post hoc test (* p<0.05, *** p<0.001).</p

Most metabolic pathways are represented in metabolites isolated from liver mitochondria.

<p>Ten pathways containing the most matches are plotted.</p

Increased endogenous mouse Trx1 level in nuclei by infection with influenza H1N1 virus.

<p>WT mice infected with influenza H1N1 virus (A/California/04/2009) at day 3 post infection (day 3) or uninfected WT control mice (day 0) were examined for nuclear Trx1. Cytoplasm (Cyto) and nuclear (Nuc) fractions of lung tissues (A) and immune cells isolated from lungs were examined for Trx1 level by western blot analysis. The same blot was probed with a lamin A/C antibody as a nuclear protein marker. Results are representatives of 3 analyses.</p

NLS-hTrx1 transgenic mice have increased NF-κB activation and proinflammatory cytokine (TNF-α and IL-6) production after influenza viral infection.

<p>Lung tissues obtained from Tg and WT before (3 mice each for Tg and WT) and 3-d post infection (6 mice for Tg, 5 mice for WT) were examined for mRNA of cytokines (A) and NF-κB activity (B). mRNA levels of TNF-α and IL-6 were analyzed and quantified by real-time PCR. * p<0.05 for 3-d post infection in Tg compared to 3 d post infection in WT and 0 d in Tg. B, NF-κB activity was examined by EMSA using same tissues analyzed for cytokines shown in A. Bands indicated as NF-κB bound DNA probe show activity of NF-κB. Densitometry values shown as fold difference were obtained from measuring relative intensities compared to that in WT before infection (lane 2). [lanes 1–6 are as follows; 1, NF-κB probe alone; 2, WT before infection; 3, Tg before infection; 4, WT 3 days post infection; 5, Tg 3-d post infection; 6, Tg 3-d post infection (cold NF-κB probe incubation followed by labeled NF-κB probe incubation)]. C, NF-κB activity (NF-κB Luc) was examined by expression of NLS-hTrx1 WT or dominant negative mutant of Trx1, NLS-hTrx1 C35S by transient cotransfection with NF-κB luciferase and β-galactosidase. Quantified luciferase activity as a measure of NF-κB activity was normalized by β-galactosidase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone.0018918-Go4" target="_blank">[37]</a>. Western blot confirms expression of NLS-hTrx1 WT and NLS-hTrx1 C35S (C, top) and β-actin (C, middle) for equal protein loading in HeLa cells 2 d after transfection. Results of luciferase activity in bar graph are shown as means ± SE (n = 8, * p<0.05).</p

Effect of influenza virus infection on (A) body weight and (B) survival in NLS-hTrx1 Tg mice compared to WT littermates.

<p>WT (n = 18) and Tg mice (n = 17) were infected with 2009 H1N1 influenza A/California/04/2009 virus (1× LD50) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone.0018918-Quan1" target="_blank">[38]</a>. Body weight and survival rates (WT; 7/18, Tg; 2/17) were monitored daily and presented as percentages based on day 0.</p

Distribution of metabolites resolved by anion exchange (A) and reverse phase (B) chromatography.

<p>To determine if a metabolite was present in the mitochondrial isolation and not a buffer contaminant, a ratio of ion intensity (sample ion intensity/buffer ion intensity) was calculated for each metabolite. A metabolite was determined to be “mitochondrial” if this ratio (s/b) was greater than or equal to 4. Additionally, employment of two chromatographic techniques resulted in the detection of 2127 mitochondrial metabolites (C).</p