Inflammatory cell counts in the lungs from mice challenged with OVA and DOK-1 shRNA.

<p>Inflammatory cell counts in the lungs from mice challenged with OVA and DOK-1 shRNA.</p

Effect of DOK-1 in OVA-induced tissue inflammation and mucus responses.

<p>(A) Lung sections were stained with hematoxylin and eosin, D-PAS, alcian blue for the evaluation of inflammatory cells and airway mucus responses. ×40 of original magnification. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression. At least 4 mice were included in each group. (B) Inflammatory index that scored parenchymal inflammation. At least 4 mice were included in each group. *P<0.05 (C) Mucus index evaluated by morphometric analysis representing alcian blue stained mucus cells (percentage of positive cells) in airway epithelial cells. At least 4 mice were included in each group. *P<0.05.</p

Effects of DOK-1 in OVA-induced inflammation and airway response.

<p>(A) The recovery of BAL cells 24 hr after OVA challenge. NEU, Neutrophil; EOS, Eosinophil; LYM, Lymphocyte; MAC, Macrophages; TOT, total cell. (B) Eosinophil peroxidase (EPO) activity in BAL fluids of OVA-sensitized and –challenged mice. (C) Airway responsiveness to aerosolized methacholine measured by non-invasive whole body plethysmography. (D) Serum IgE and IgG2a levels detected by ELISA. The values in all the panels represent means ± S.E.M. At least 5 mice were included in each group. *P<0.05, ***P<0.001 vs. OVA-challenged mice.</p

Effects of DOK-1 on STAT-6 and STAT-4 expression and nuclear translocation in airway epithelial cells.

<p>(A) Lung sections were stained with Alexa Fluor 488-conjugated DOK-1 and Alexa Fluor 568-conjugated STAT-6 antibodies and DAPI stain. (B) Lung sections were stained with Alexa Fluor 488-conjugated DOK-1 and Alexa Fluor 568-conjugated STAT-4 antibodies and DAPI stains. A representative photo of 7 similar experiments. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression.</p

DOK-1 expression in the lungs after OVA sensitization and challenge.

<p>The lentiviral vectors containing DOK-1 specific ShRNA (DOK_ShRNA) or DOK-1 cDNA (DOK) were administrated together with controls (empty vector or vector containing non-specific scrambled ShRNA) before OVA challenge. (A) Representative western blotting demonstrating DOK-1 protein expression in the lungs of the mice. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression (B) Immunofluorescent staining on the tissue sections from the lungs using Alexa Fluor 488-conjugated DOK-1 antibody and DAPI stains.</p

Effect of DOK-1 on STAT-6 and STAT-4 signaling pathways.

<p>Expression and/or activation (phosphorylation) of STAT6, STAT4, GATA3, and T-bet transcriptional factors were evaluated by Western blot analysis. A representative gel photo out of 6 similar independent experiments. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression.</p

Effect of DOK-1 on T cells and cytokine/chemokine expression.

<p>(A) FACS Histogram analysis on BAL cells from the mice after OVA sensitization and challenge. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression. Each lane indicates CD4⁺ T cell population stained with Cy5-anti-CD4 antibody. (B) BAL CD4(+)T cells gated with PE-conjugated CD4 were further evaluated by intracellular staining against Cy5-conjugated-IL-4 and FITC-conjugated-IFN-γ. (C) The levels of inflammatory Th1 and Th2 cytokines and a chemokine in BAL fluid were measured by ELISA at 24 hrs after the last OVA challenge. Data represent means ± S.E.M. Each group contains at least 7 mice. *P<0.05, ***P<0.001 vs. OVA-challenged mice.</p