Journal:

Article Title: Hepatocyte nuclear factor 4? orchestrates expression of cell adhesion proteins during the epithelial transformation of the developing liver

doi: 10.1073/pnas.0600246103

Figure Lengend Snippet: Hepatocyte-specific loss of E-cadherin does not affect the formation of cell junctions in the liver. (A) RT-PCR showed loss of E-cadherin (E-cad) mRNA in livers of Cdh1loxP/loxP;AlfpCre mice compared with control Cdh1loxP/+;AlfpCre and WT littermates. Hnf4a levels were unchanged, and hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) confirmed equal loading. (B) Immunoblot analysis of liver extracts indicated that E-cadherin (E-cad) protein is undetectable in the Cdh1loxP/loxP;AlfpCre livers compared with controls (Cdh1loxP/+;AlfpCre and WT). Total protein levels of the tight junction protein OCLN were unchanged, and β-actin (ACTB) demonstrated equal loading. (C) Immunohistochemistry detected E-cadherin between hepatocytes in control livers (Top Left) but not between hepatocytes in Cdh1loxP/loxP;AlfpCre livers (Top Right) (Inset is higher magnification). Confocal immunofluorescence microscopy was used to detect TJP1 (also known as ZO1) at the apical surface of the hepatocytes in both control (Cdh1loxP/+;AlfpCre, Middle Left) and Cdh1loxP/loxP;AlfpCre (Middle Right) livers. Junctional complexes (indicated by brackets) were identified in both control (Cdh1loxP/+;AlfpCre, Bottom Left) and Cdh1loxP/loxP;AlfpCre (Bottom Right) livers by transmission electron microscopy. Asterisks indicate bile canaliculi, which confirm that hepatocytes are polarized in the absence of E-cadherin. High-resolution electron microscopy images are provided in Fig. 5, which is published as supporting information on the PNAS web site.

Article Snippet: Antibodies against the following proteins were used: claudin-1 (rabbit polyclonal; Zymed; 1:100), connexin 32 (rabbit polyclonal; Zymed; 1:200), ZO1 (rabbit polyclonal; Zymed; 1:200), and Alexa-Fluor 488 goat anti-rabbit (Invitrogen; 1:500).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Immunofluorescence, Microscopy, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Adaptive remodeling of rat adrenomedullary stimulus-secretion coupling in response to a chronic hypertensive environment

doi: 10.1101/2023.11.28.568973

Figure Lengend Snippet: A. Reduced Lucifer yellow (LY) diffusion between SHR chromaffin cells. LY was introduced into chromaffin cells using patch pipettes. Dye diffusion was imaged 15 minutes after patch disruption. Less than 20% of SHR chromaffin cells are dye-coupled, as compared to more than 30% in WKY rats. B. Decreased expression level of Gja1 (encoding Cx43), but not Gjd2 (encoding Cx36) in SHRs, assessed by real-time RT-PCR from macrodissected medullary tissues. C. Western blots and associated pooled data histograms of Cx43 and ZO-1, an associated protein. Cx43 expression, but not ZO-1, is significantly down-regulated in SHRs.

Article Snippet: Blots were next incubated with polyclonal antibodies, a rabbit anti-Cx43 (1:500, germo Fisher Scientific) or a rabbit anti-ZO-1 (1:250, germo Fisher Scientific) in TBS-Tween 0.1% containing 5% milk, at 4°C overnight.

Techniques: Diffusion-based Assay, Disruption, Expressing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Phthalate monoesters affect membrane fluidity and cell-cell contacts in endometrial stromal cell lines

doi: 10.1101/2024.06.17.599271

Figure Lengend Snippet: ZO-1 immunostaining in T-HESC spheroids reveals that MEHHP interferes with tight junction assembly. A and B, deconvolved fluorescence microscopy images from a single representative experiment showing the overlay of ZO-1 in the green channel and nuclear stain in the blue channel (10× objective). The compounds used for 72-h treatment are indicated in the top right (A: 0.1% DMSO, B: 21.6 μM MEHHP) and the scale bar is shown in the bottom left corner in A. C, quantification of ZO-1 average immunostaining intensity per spheroid area unit (pooled data, 2-3 spheroids per experiment, N = 3); circles denote individual spheroids, the middle line shows the median, and the whiskers protrude from the second to the third quartile. To compensate for the variation in staining conditions between the experiments, for each independent experiment, the ZO-1 signal was normalized to the mean signal observed in spheroids treated with 0.1% DMSO. The asterisks indicate the statistical significance of difference between the ZO-1 signal in the absence vs presence of MEHHP; ** corresponds to P < 0.01.

Article Snippet: The immunostaining of tight junction proteins was conducted using rabbit monoclonal antibody against human Zonula Occludens 1 protein (ZO-1; Abcam ab221547, UK), rabbit monoclonal antibody against human Occludin (Abcam ab222691, UK), or rabbit monoclonal antibody against human Claudin 1 (Abcam, ab15098, UK).

Techniques: Immunostaining, Fluorescence, Microscopy, Staining

Journal:

Article Title: Establishment of Systemic Brucella melitensis Infection through the Digestive Tract Requires Urease, the Type IV Secretion System, and Lipopolysaccharide O Antigen ▿

doi: 10.1128/IAI.00417-09

Figure Lengend Snippet: Interaction of B. melitensis with polarized Caco-2 enterocyte monolayers and Caco-2 cells with M-like cells. (A) Transepithelial electrical resistance was measured before infection of monolayers and 2 h after infection. Data shown are from individual experiments that are representative of at least three experimental replicates. The statistical significance of differences in the values for the experimental groups was assessed using Student's t test. Values that are statistically significantly different are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Distribution of the tight-junction marker ZO-1 in monolayers after infection with B. melitensis. The large cells in the bottom panels have the characteristic large size of M cells. DAPI, 4′,6′-diamidino-2-phenylindole.

Article Snippet: The samples were washed three times in PBS with agitation and incubated for 60 min with rabbit anti-ZO-1 (ZO-1 is zona occludens 1) (1:40, Invitrogen) and 2% goat serum in PBS.

Techniques: Infection, Marker