bDNA-RNA versus RT-PCR.

<p>bDNA-RNA versus RT-PCR.</p

Validation analysis between bDNA-RNA and bDNA-lysate.

<p>Data of bDNA-RNA and bDNA-lysate were plotted against each other. The expression levels of genes were measured using purified RNA by bDNA-RNA, and using lysate by bDNA-lysate. Data of bDNA-RNA were from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124392#pone.0124392.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124392#pone.0124392.g003" target="_blank">3</a>. Expression levels were shown relative to β-actin.</p

bDNA-lysate versus RT-PCR.

<p>bDNA-lysate versus RT-PCR.</p

Evaluation of seasonal influenza vaccine with QGP.

<p>The relative gene expression levels of the <i>Gapdh</i> gene are indicated in each column (grades 1, 2 and 3, respectively). *Significant difference between B and C. **Significant difference between B, C and D, ***Significant difference between PD and WPv.</p

Expression of Albatross and desmoplakins in the thymi of keratin 8-deficient mice.

<p>Immunofluorescence staining of thymus sections of 7-week-old wild-type FVB/N mice (WT) and K8-deficient FVB/N mice (K8 KO) was performed to detect CD205 (<b>A</b>) or desmoplakins I/II (<b>C</b>) (green) and Albatross (<b>A</b>) (red). (<b>A</b>) Whereas colocalization of CD205 and Albatross is typically seen in wild-type, Albatross expression was reduced in the cortex of K8-deficient thymus. (<b>B</b>) Semi-quantitative RT-PCR analysis for Albatross was performed on Total RNA obtained from each thymus: <i>lower panel</i>, equal loading as defined by the levels of β-actin mRNA. (<b>C</b>) Localization of desmoplakins I/II is unaltered in K8 deficient thymus. Data are representative of independent experiments (<i>n = </i>8 in each group). C, cortex; M, medulla. Scale bars = 100 µm.</p

Keratin 8-deficient mice are absent from keratin 8/18 in thymic cortex.

<p>Immunofluorescence staining of thymus sections of 7-week-old wild-type FVB/N mice (WT) and K8-deficient FVB/N mice (K8 KO) was performed to detect K8 (<b>A</b>) or K18 (<b>B</b>) (green) and K5 (<b>A</b>) or K14 (<b>B</b>) (red). K8-deficient mice are lack of K8/K18 expression in the cortex. Note the altered morphology of K5+ or K14+ mTECs in K8-deficient mice. Data are representative of independent experiments (<i>n = </i>8 in each group). C, cortex; M, medulla. Scale bars = 100 µm. (<b>C</b>) Thymi from wild-type FVB/N mice and K8-deficient FVB/N mice at 5 weeks and 9 weeks of age were weighed. Each point provides the thymus weight of individual animal.</p

Gene expression analysis of RE treated rats.

<p>One lung lobe was obtained from rats treated with various concentrations of RE in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124392#pone.0124392.g001" target="_blank">Fig 1</a>, and expression levels of genes were analyzed by bDNA-RNA, and were shown relative to β-actin. Three rats per group were used. Values are expressed as mean ± S.D. * P < 0.05, ** P < 0.01, *** P < 0.001. † P < 0.05 versus SA, †† P < 0.01 versus SA, ††† P < 0.001 versus SA.</p

Animal test and gene expression analysis of HA-v vaccine treated rats.

<p>The effects of HA-v were measured using ATT, LTT, PLT counts, and gene expression analysis. All rats were analyzed at day 1 after vaccine injection. Four rats were used for HA, and three rats were used for HA-v. (A) Changes in rat body weight were assessed as the percentage increase or decrease, and are indicated by the mean change ± S.D. (B, C) WBC and PLT of peripheral blood were measured and plotted in graphs. Each symbol corresponds to an individual rat. Difference in symbol colors represents different batches of HA. (D) The expression levels of genes were analyzed by bDNA-lysate, and were shown relative to β-actin. Values are expressed as mean ± S.D. * P < 0.05, ** P < 0.01, *** P < 0.001.</p

Summary of biomarker studies.

<p>Biomarkers used in this study were strongly correlated with immune responses after influenza infection.</p

Abnormal toxicity test (ATT) and hematological tests of influenza vaccine-treated rats.

<p>The effects of RE, HA bulk materials, and SA treatment were measured using ATT, LTT and platelet (PLT) counts. Three rats per group were used, and were analyzed at day 1 after vaccine administration. (A) Changes in rat body weight were assessed as the percentage increase or decrease, and are indicated by the mean change ± S.D. WBC (B) and PLT (C) of peripheral blood were measured and plotted. Each symbol corresponds to an individual rat. Symbols filled with a few spots, a medium amount of spots, and many spots indicate 0.25, 0.5, and 1 U/ml of RE. Pale gray, gray, and black in each symbol indicate 1x, 2x, and 4x concentrated HA bulk materials. One blood sample of HA-A (2x) was clotted and omitted from LTT and PLT counts. * P < 0.05, ** P < 0.01. † P < 0.05 versus SA, †† P < 0.01 versus SA, ††† P < 0.001 versus SA.</p