Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection

doi: 10.1152/ajplung.00345.2018

Figure Lengend Snippet: Respiratory syncytial virus (RSV) infection disrupts the integrity of airway epithelial junctions. Lung tissue sections of control, UV-RSV, and live RSV-infected animals were subjected to immunofluorescence labeling for tight-junction (TJ) proteins [zonula occludens-1 (ZO-1), occludin, claudin-1, claudin-2, claudin-18) and adherens junctions (AJ) (β-catenin, E-cadherin) proteins. Labeled sections were examined by confocal microscopy. Arrows indicate intact junctional complexes in airways of control animals. Arrowheads point to disrupted TJ structure and increased claudin-2 labeling in tissue sections of RSV-infected mice. Scale bar, 40 μm. Images are representative of ≥3 experiments, with >5 mice per group.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect junctional and signaling proteins by immunofluorescence labeling and immunoblotting: anti-occludin mAb, anti-ZO-1 pAb, anti-claudin-18 mAb (Thermo-Fisher Scientific, Waltham, MA); anti-claudin-1 pAb, anti-claudin-2 pAb, anti-GAPDH mAb (Abcam, Cambridge, MA); anti-β-catenin mAb, anti-E-cadherin mAb (BD Bioscience, San Jose, CA); and anti-soluble E-cadherin mAb (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Infection, Immunofluorescence, Labeling, Confocal Microscopy

Journal: Communications Biology

Article Title: Hepatocyte growth factor activator inhibitor-2 stabilizes Epcam and maintains epithelial organization in the mouse intestine

doi: 10.1038/s42003-018-0255-8

Figure Lengend Snippet: Effects of Spint2 deletion on the gastrointestinal tract. a Decreased intestinal length in Spint2 -deleted mice. Representative macroscopic photos of gastrointestinal tissue (left; bars, 2 cm) and box plots of intestinal length (right) are shown. 1, Spint2 LoxP/LoxP mice treated with tamoxifen (Tam); 2, Spint2 LoxP/LoxP CreERT2 mice treated with corn oil only (vehicle); 3, Spint2 LoxP/+ CreERT2 mice treated with Tam; 4, Spint2 LoxP/LoxP CreERT2 mice treated with Tam. The 25th and 75th percentile (boxes) and the median (bold line within the boxes) are plotted. Circle represents the value of each case. N = 8 for each group. p- value; Mann–Whitney U- test. b Histology of small intestine ( Spint2 LoxP/LoxP CreERT2 mice) tissue 72 h after tamoxifen or vehicle treatment. Representative photos of HE-stained sections from the proximal small intestine of the vehicle-treated mouse (upper panel, left), proximal small intestine of the tamoxifen-treated mouse (center) and distal small intestine of the tamoxifen-treated mouse (right) are shown. Arrow indicates a tuft formed by enterocytes. Photos of Hai-2 and Ki67 immunohistochemistry are also shown in the lower panel. Bars, 50 μm. c Detachment of surface epithelial sheets observed 48 h after tamoxifen treatment. Representative photos of HE-stained and cleaved caspase-3 (cCas3)-stained section from the proximal small intestine (left panel) are shown. Distal small intestine showed similar change and the cells were also immunohistochemically positive for ssDNA (right upper panel). At 72 h after tamoxifen treatment, villi were shortened with many cCas3-positive cells in the upper portion and increased numbers of apoptotic bodies in the crypts (proximal small intestine; right lower panel). Bars, 50 μm. d Decreased numbers of Paneth cells in the small intestine. HE staining and immunohistochemical staining for lysozyme are shown. Bars, 50 μm

Article Snippet: The following anti-mouse antibodies were used: anti-Hai-2 goat polyclonal antibody (pAb) (R & D Systems, Minneapolis, MN); anti-Epcam (ProteinTech., Rosemont, IL), anti-claudin-7 (Thermo Fisher Scientific, Waltham, MA), anti-mouse β-catenin (Sigma, St. Louis, MO), anti-cleaved caspase-3 (Cell Signaling, Boston, MA), and anti-lysozyme (AVIVA Systems Biology, San Diego, CA) rabbit pAbs; anti-Ki67 rabbit monoclonal antibody (mAb) (Abcam, Cambridge, UK); anti-E-cadherin goat pAb (R & D Systems); anti-claudin-2 and anti-ZO-1 rabbit pAbs (Thermo Fisher Scientific); anti-β-actin mouse mAb (Sigma) and anti-ssDNA rabbit pAb (Dako, Carpinteria, CA).

Techniques: MANN-WHITNEY, Staining, Immunohistochemistry, Immunohistochemical staining

Journal: Communications Biology

Article Title: Hepatocyte growth factor activator inhibitor-2 stabilizes Epcam and maintains epithelial organization in the mouse intestine

doi: 10.1038/s42003-018-0255-8

Figure Lengend Snippet: Effects of Hai-2 ablation on large intestine. a Histology of the cecum from Spint2 LoxP/LoxP CreERT2 mice after tamoxifen or vehicle (corn oil) treatment. Representative photos of HE from mice 24, 36, and 72 h after tamoxifen treatment are shown. Successful Hai-2 ablation was confirmed by immunohistochemistry. b Histology of the colon 72 h after tamoxifen treatment. Crypts were elongated with many exfoliating, degenerated cells present in the lumen. Arrows indicate a tuft formed by enterocytes. Higher magnification images are shown in the insets. Representative photos of Ki67 immunohistochemistry are also presented (right panel). c Decreased numbers of goblet cell in Spint2 -deleted colon mucosa. Alcian-blue staining. The 25th and 75th percentile (boxes) and the median (bold line within the boxes) are plotted. Circle represents the value of each case. Goblet cells per crypt of Spint2 -deleted mice ( n = 5 and 11 for 48 h and 72 h after starting treatment, respectively) in the proximal and distal colon were counted and compared to that for vehicle-treated control mice ( n = 8). p- value; Mann–Whitney U- test. Bars, 50 μm ( a – c )

Article Snippet: The following anti-mouse antibodies were used: anti-Hai-2 goat polyclonal antibody (pAb) (R & D Systems, Minneapolis, MN); anti-Epcam (ProteinTech., Rosemont, IL), anti-claudin-7 (Thermo Fisher Scientific, Waltham, MA), anti-mouse β-catenin (Sigma, St. Louis, MO), anti-cleaved caspase-3 (Cell Signaling, Boston, MA), and anti-lysozyme (AVIVA Systems Biology, San Diego, CA) rabbit pAbs; anti-Ki67 rabbit monoclonal antibody (mAb) (Abcam, Cambridge, UK); anti-E-cadherin goat pAb (R & D Systems); anti-claudin-2 and anti-ZO-1 rabbit pAbs (Thermo Fisher Scientific); anti-β-actin mouse mAb (Sigma) and anti-ssDNA rabbit pAb (Dako, Carpinteria, CA).

Techniques: Immunohistochemistry, Staining, MANN-WHITNEY

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection

doi: 10.1152/ajplung.00345.2018

Figure Lengend Snippet: Respiratory syncytial virus (RSV) infection disrupts the integrity of airway epithelial junctions. Lung tissue sections of control, UV-RSV, and live RSV-infected animals were subjected to immunofluorescence labeling for tight-junction (TJ) proteins [zonula occludens-1 (ZO-1), occludin, claudin-1, claudin-2, claudin-18) and adherens junctions (AJ) (β-catenin, E-cadherin) proteins. Labeled sections were examined by confocal microscopy. Arrows indicate intact junctional complexes in airways of control animals. Arrowheads point to disrupted TJ structure and increased claudin-2 labeling in tissue sections of RSV-infected mice. Scale bar, 40 μm. Images are representative of ≥3 experiments, with >5 mice per group.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect junctional and signaling proteins by immunofluorescence labeling and immunoblotting: anti-occludin mAb, anti-ZO-1 pAb, anti-claudin-18 mAb (Thermo-Fisher Scientific, Waltham, MA); anti-claudin-1 pAb, anti-claudin-2 pAb, anti-GAPDH mAb (Abcam, Cambridge, MA); anti-β-catenin mAb, anti-E-cadherin mAb (BD Bioscience, San Jose, CA); and anti-soluble E-cadherin mAb (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Virus, Infection, Control, Immunofluorescence, Labeling, Confocal Microscopy

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection

doi: 10.1152/ajplung.00345.2018

Figure Lengend Snippet: Respiratory syncytial virus (RSV) infection induces accumulation of soluble E-cadherin in the bronchoalveolar lavage (BAL). BAL fluid collected from control, UV-RSV, and live RSV-infected mice (A and B) as well as cell culture medium collected from control, UV-RSV, and live RSV-infected mouse tracheal epithelial cell monolayers (mTEC; C) were analyzed for the presence of soluble E-cadherin fragments. Representative immunoblot images (A and C) and densitometric quantification of protein band intensity (B) are shown. D: recombinant extracellular E-cadherin fragment (10 µg/ml) was added to nonconfluent human primary airway epithelial cells. Cells were immunofluorescently stained for tight-junction (TJ) protein zonula occludens-1 (ZO-1) after 4 days. Scale bar, 40 μm. Data are presented as means ± SE, n ≥ 3. ***P < 0.001.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect junctional and signaling proteins by immunofluorescence labeling and immunoblotting: anti-occludin mAb, anti-ZO-1 pAb, anti-claudin-18 mAb (Thermo-Fisher Scientific, Waltham, MA); anti-claudin-1 pAb, anti-claudin-2 pAb, anti-GAPDH mAb (Abcam, Cambridge, MA); anti-β-catenin mAb, anti-E-cadherin mAb (BD Bioscience, San Jose, CA); and anti-soluble E-cadherin mAb (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Virus, Infection, Control, Cell Culture, Western Blot, Recombinant, Staining