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: mBio

Article Title: Zika Virus NS2A-Mediated Virion Assembly

doi: 10.1128/mBio.02375-19

Figure Lengend Snippet: NS2A interacts with prM, E, NS2B, and NS3 proteins in HEK293T cells. HEK293T cells were transfected with two plasmids, one encoding HA-NS2A and the other encoding Flag-tagged prM (A), E (B), NS2B (C), or NS3 (D). At 24 h p.t., cell lysates were precipitated with HA or Flag antibodies. Viral proteins in the cell lysates and IP eluates were detected by Western blotting using corresponding antibodies.

Article Snippet: The following antibodies were used in this study: mouse monoclonal antibody 4G2 cross-reactive with flavivirus E protein (ATCC); rabbit anti-E polyclonal antibody (GeneTex); rabbit anti-HA monoclonal antibody (Cell Signaling Technology); mouse anti-HA monoclonal antibody (Cell Signaling Technology); rabbit anti-Flag polyclonal antibody (Cell Signaling Technology); mouse anticapsid antiserum (generated in-house); rabbit anti-prM polyclonal antibody (GeneTex); rabbit anti-NS1 polyclonal antibody (GeneTex); rabbit anti-NS3 polyclonal antibody (GeneTex); mouse monoclonal antibody cross-reactive with ZIKV and DENV-2 NS4B (clone 44-4-7) ( ); mouse anti-NS5 antiserum (generated in-house); mouse anti-dsRNA monoclonal antibody, clone J2 (SCICONS English & Scientific Consulting Kft); rabbit anticalnexin polyclonal antibody (Sigma-Aldrich); rabbit anti-GM130 monoclonal antibody (Cell Signaling Technology); rabbit anti-NS2B polyclonal antibody (GeneTex); rabbit anti-GAPDH polyclonal antibody (Sigma-Aldrich); rabbit anti-claudin-2 polyclonal antibody (Invitrogen); rabbit anti-GST polyclonal antibody (Abcam); goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 568 (ThermoFisher Scientific); goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific); goat anti-rabbit IgG polyclonal antibody conjugated with Alexa Fluor 568 (ThermoFisher Scientific); goat anti-rabbit IgG polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific); goat anti-mouse and anti-rabbit IgG polyclonal antibodies conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich); protein A conjugated with HRP (Sigma-Aldrich); rabbit IgG isotype control (Invitrogen); and mouse IgG isotype control (Invitrogen).

Techniques: Transfection, Western Blot

Journal: mBio

Article Title: Zika Virus NS2A-Mediated Virion Assembly

doi: 10.1128/mBio.02375-19

Figure Lengend Snippet: NS2A E103A disrupts the association between prM/E complex and NS2B/NS3 complex. (A) Diagram of the hypothesized interaction network among NS2A, prM/E, and NS2B/3 complexes. Dotted boxes indicate prM/E and NS2B/NS3 complexes. Lines with two arrows indicate protein-protein interactions. Red lines indicate loss of interactions. (B) Co-IP analysis of genomic ZIKV RNA-transfected cells. BHK-21 cells (8 × 10 6 ) were electroporated with 2 μg of HA-NS2A ZIKV RNA or 15 μg E103A HA-NS2A ZIKV RNA. The different amounts of RNAs were used to achieve comparable expression levels of viral proteins in HA-NS2A ZIKV- and E103A HA-NS2A ZIKV-transfected cells upon sample collection. Untransfected BHK-21 cells (Mock) were used as control. At 24 h p.t., cells were lysed and subjected to precipitation using prM antibody, NS2B antibody, or isotype control IgG. Viral proteins in the cell lysates and IP eluates were analyzed by Western blotting using corresponding antibodies. (C) Summary of the network among NS2A, prM, E, NS2B, and NS3 proteins. The left panel shows that the WT NS2A acts as an intermediator of prM/E and NS2B/3 complexes. The right panel presents the dissociation between prM/E and NS2B/3 complexes due to the NS2A E103A mutation.

Article Snippet: The following antibodies were used in this study: mouse monoclonal antibody 4G2 cross-reactive with flavivirus E protein (ATCC); rabbit anti-E polyclonal antibody (GeneTex); rabbit anti-HA monoclonal antibody (Cell Signaling Technology); mouse anti-HA monoclonal antibody (Cell Signaling Technology); rabbit anti-Flag polyclonal antibody (Cell Signaling Technology); mouse anticapsid antiserum (generated in-house); rabbit anti-prM polyclonal antibody (GeneTex); rabbit anti-NS1 polyclonal antibody (GeneTex); rabbit anti-NS3 polyclonal antibody (GeneTex); mouse monoclonal antibody cross-reactive with ZIKV and DENV-2 NS4B (clone 44-4-7) ( ); mouse anti-NS5 antiserum (generated in-house); mouse anti-dsRNA monoclonal antibody, clone J2 (SCICONS English & Scientific Consulting Kft); rabbit anticalnexin polyclonal antibody (Sigma-Aldrich); rabbit anti-GM130 monoclonal antibody (Cell Signaling Technology); rabbit anti-NS2B polyclonal antibody (GeneTex); rabbit anti-GAPDH polyclonal antibody (Sigma-Aldrich); rabbit anti-claudin-2 polyclonal antibody (Invitrogen); rabbit anti-GST polyclonal antibody (Abcam); goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 568 (ThermoFisher Scientific); goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific); goat anti-rabbit IgG polyclonal antibody conjugated with Alexa Fluor 568 (ThermoFisher Scientific); goat anti-rabbit IgG polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific); goat anti-mouse and anti-rabbit IgG polyclonal antibodies conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich); protein A conjugated with HRP (Sigma-Aldrich); rabbit IgG isotype control (Invitrogen); and mouse IgG isotype control (Invitrogen).

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Expressing, Control, Western Blot, Mutagenesis