Journal:

Article Title: Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease

doi: 10.1073/pnas.0504787102

Figure Lengend Snippet: Dysregulated expression of CCR7 in Runx3 KO DC. (A) Impaired TGFβ-dependent inhibition of CCR7 transcription in KO BMDC. WT and Runx3 KO BMDC were grown in the presence or absence of TGFβ (10 ng/ml). At day 6, cells were induced to undergo maturation by LPS, and, at day 7, RNA was prepared and analyzed by RT-PCR. (B) Impaired TGFβ-dependent inhibition of surface expression of CCR7 in KO BMDC. WT and Runx3 KO BMDC were grown and treated as in A. At day 7, cells were analyzed by FACS by using anti-CCR7 antibodies (goat anti-mouse CI0131, Capralogics). FSChighCD11c+ DC were gated, and their CCR7 expression was determined. Reduction in the level of surface CCR7 and in the number of cells expressing it was noted only in WT and not in Runx3 KO BMDC. (C and D) Increased CCR7 expression on alveolar and LN DC of Runx3 KO mice. BAL and peripheral LN cells of KO and WT mice (n = 3) were obtained and analyzed. (C) Alveolar DC (FSChigh/CD11chigh) were gated and analyzed for CCR7 expression. Of note, expression of CCR7 on the DC subpopulation CD11c+/CD11b+ present only in KO lungs is shown along with that of KO CD11c+/CD11b- DC. (D) FSChigh/CD11chigh DC of axillary and thoracic LN were gated and analyzed for CCR7 expression.

Article Snippet: At day 7, cells were analyzed by FACS by using anti-CCR7 antibodies (goat anti-mouse CI0131, Capralogics).

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease

doi: 10.1073/pnas.0504787102

Figure Lengend Snippet: Elevated CCR7 mediated in vivo trafficking of alveolar DC to the draining LN in the KO mice. (A and B) Runx3 KO (n = 7) and WT (n = 5) mice were treated by intranasal administration of CFSE to label in vivo the respiratory DC. When indicated, WT mice were treated with LPS (n = 4), and Runx3 KO mice were treated with anti-CCR7 antibody (n = 4) or with buffer only (n = 4). KO mice (n = 4) and LPS-treated WT mice (n = 3) were also treated by inhalation of Ciglitazone. Eighteen hours later, mice were killed, and single-cell suspensions of BAL, thoracic LN, and axillary LN were prepared and analyzed by FACS. (A) FSChigh/CD11c+ DC were gated (R1 and R2). Shown is representative side scatter (SSC) versus CFSE staining of DC populations in BAL and LN after the various treatments. (B) Migration index of alveolar DC to thoracic LN represents the ratio between the percentage of CFSE+ cells within the CD11c+ population in the thoracic LN and the respective value in BAL cells. Results are presented as mean ± SEM. Analysis of variance showed that the migration index of untreated KO DC was significantly higher than that of WT (*, P = 0.016). Notably, the anti-CCR7-treated KO DC migration index was similar to basal migration of WT, and Ciglitazone significantly (*, P = 0.03) reduced the migration of KO DC.

Article Snippet: At day 7, cells were analyzed by FACS by using anti-CCR7 antibodies (goat anti-mouse CI0131, Capralogics).

Techniques: In Vivo, Staining, Migration

Journal: The Journal of Cell Biology

Article Title: Effects of Forced Expression of an NH 2 -terminal Truncated β-Catenin on Mouse Intestinal Epithelial Homeostasis

doi:

Figure Lengend Snippet: Forced expression of ΔN89β-catenin has no discernible effects on epithelial cell differentiation. Frozen sections were prepared from PLP-fixed jejunums of 6-mo-old chimeric-transgenic animals. ( A ) A polyclonal villus stained with biotin-conjugated Dolichos biflorus agglutinin ( DBA ), Cy3-conjugated avidin, rabbit anti–β-gal, and FITC-conjugated donkey anti–rabbit Ig. Glycoconjugates containing GalNAcα3GalNAc and GalNAcα3Gal recognized by DBA appear yellow-orange; β-gal appears green. The polarity and differentiation of enterocytes appears to be unaffected, as judged by the distribution of these glycoconjugates in apical membranes and supranuclear Golgi apparatus ( closed arrow ). Similarly, based on their reaction with DBA, the number and differentiation of goblet cells ( open arrows ) is equivalent in the 129/Sv and B6-ROSA26 components of the polyclonal villus. ( B ) The base of a polyclonal villus with its crypt-villus junction indicated by closed arrows . Three crypts are seen ( open arrows at their base): the one on the left is supplying cells to another villus. The section was incubated with rat anti-β 4 integrin subunit, Cy3 donkey anti–rat Ig, rabbit anti–β-gal, FITC donkey anti–rabbit Ig, and bis-benzimide. Nuclei ( blue ); the β 4 integrin subunit ( orange ); β-gal ( green-brown ). The location of β 4 integrin at the base of epithelial cells and its distribution along the crypt-villus unit are unaffected by ΔN89β-catenin. ( C ) Villi sectioned perpendicular to their crypt-villus axis. The tight junction protein ZO-1 ( orange ) was detected with rat anti-ZO-1 and Cy3 donkey anti–rat Ig. β-Gal ( green ) was visualized with the same reagents used in the preceding sections. The levels and location of ZO-1 in the 129/Sv(ΔN89β-catenin) and B6-ROSA26 components of polyclonal villi are similar (e.g., open arrows ). ( D ) Villi sectioned perpendicular to their crypt-villus axis as in C . The section was incubated with rat anti-β 7 integrin, Cy3-donkey anti–rat Ig, rabbit anti-laminin, rabbit anti–β-gal, FITC donkey anti–rabbit Ig, and bis-benzimide. B6-ROSA26 cells exhibit diffuse staining of their cytoplasm due to the presence of β-gal ( green ). β 7 integrin is confined to intraepithelial lymphocytes ( orange ). Comparable numbers of these cells are seen in the B6-ROSA26 and 129/ Sv(ΔN89β-catenin) components of polyclonal villi and in wholly 129/Sv(ΔN89β-catenin) villi. Laminin appears as linear green immunoreactivity underlying 129/Sv and B6-ROSA26 epithelium (e.g., closed arrows ). The intensity of staining is similar under cells of both genotypes. Bars, 25 μm.

Article Snippet: PLP-fixed frozen sections of jejunum were stained with a 19-member panel of antibodies: ( a ) affinity-purified rabbit anti– Escherichia coli β-galactosidase (β-gal) (1:500; 5′→ 3′ Inc., Boulder, CO); ( b ) rabbit anti– β-catenin sera (see above, final dilution in PBS/blocking buffer = 1:500); ( c ) affinity-purified rabbit anti–c-myc (see above, 1:100); ( d ) affinity-purified rabbit antibodies raised against amino acids 1034–2130 of human APC (APC2, a gift of P. Polakis; ; ); ( e ) affinity-purified rabbit anti–α-catenin (1:500; gift of J. Nelson); ( f ) a monoclonal rat antibody to E-cadherin (1:1,000; Sigma Chemical Co. ; Hermiston et al., 1995 a ); ( g ) rat anti–ZO-1 (polyclonal antibodies, 1:50; Chemicon International, Inc., Temecula, CA); ( h ) rabbit anti-laminin (1: 1,000; Chemicon International Inc.); ( i ) rabbit anti-mouse fibronectin (1: 1,000; Chemicon International Inc.); ( j ) rabbit anti-mouse collagen type IV (1:1,000; Chemicon International Inc.); ( k ) rat anti-mouse β 1 integrin (1:500; PharMingen , San Diego, CA); ( l ) rat anti-mouse β 7 integrin (1:500; PharMingen ); ( m ) rat anti-mouse β 4 integrin (1:500; PharMingen ); ( n ) rat anti-mouse α 6 integrin (1:500; PharMingen ); ( o ) goat anti-BrdU (1:1,000; ); ( p ) rabbit anti-serotonin (1:1,000, a marker of the predominant enteroendocrine subpopulation in the adult mouse intestine; Incstar, Stillwater, MN); ( q ) rabbit anti-chromagranin A (1:1,000, a general marker of enteroendocrine cells; Incstar); and ( r ) rabbit anti-liver fatty acid binding protein (1:1,000, an enterocyte lineage marker; ).

Techniques: Expressing, Cell Differentiation, Transgenic Assay, Staining, Avidin-Biotin Assay, Incubation