Pharmacological inhibition and/or siRNA silencing of EP2 receptor in EP2S cells decrease claudin-4 expression.

<p>(A) EP2S cells that reached confluence on regular culture plates were exposed with 50 µM of AH6809 (EP2 antagonist) for the time indicated and the cell lysate analyzed for claudin-4 expression by western blotting. Actin used as the internal control for the densitometry analysis. (B) EP2S cells seeded on culture plates that reached 60 percent confluence were transfected with either EP2 receptor or control siRNA. Cells collected after 48 h were checked for claudin-4 expression by western blotting and quantified by densitometry analysis for claudin-4 expression. ★ p<0.05; ★★ p<0.01 and ★★★ p<0.001.</p

The expression of tight junction mRNA and proteins in Caco-2 EP2 receptor transfectants.

<p>(A) The mRNA expression for tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The expression of gene of interest (GOI) was normalized with the internal control (Actin) and expressed as fold changes with that of the vector control. (B) A representative immunoblot showing constitutive expression of the important tight junction proteins (ZO-1, occludin, claudin-1, claudin-2 and claudin-4) in EP2S, EP2A and vector control cells. Actin used as internal control. (C) Densitometry analysis for claudin-4 expression in EP2S, EP2A and vector control cells. EP2A compared statistically with the vector control. ★★ p<0.01.</p

Stimulation of Caco-2 cells with IFN-γ decreases claudin-4 expression.

<p>(A) Caco-2 cells grown to confluence on regular culture plates were stimulated with 5 ng/ml of IFN-γ for 48 h. At the end of stimulation, cells were analyzed for claudin-4 expression by western blotting. (B) The corresponding densitometry analysis for claudin-4 expression. (C) Western blots depicting dose and time response of IFN-γ treatment on claudin-4 expression in vector control, EP2S and EP2A cells. Three different doses of IFN-γ (1, 5 and 10 ng) exposed for 24 and 48 h time period were studied and compared with corresponding untreated control cells. V = vector control; S = EP2S; A = EP2A.</p

EP2A cells over express IFN-γ.

<p>(A) The expression of transcript for cytokines (IL-8, IL-1β, TNF-α and IFN-γ) in Caco-2 EP2S and EP2A cells was measured by Q-PCR. The gene of interest (GOI) was normalized with the internal control (Actin) and depicted as fold changes with that of vector control. IFN-γ expression in EP2A cells was statistically compared with that of vector control. (B) Cell culture supernatant collected from Caco-2 cells (vector control, EP2S and EP2A) were quantified for IFN-γ using eBioscience's Human IFN-γ ELISA Kit. ★ p<0.05; ★★ p<0.01.</p

Claudin-4 undergoes increased proteosomal degradation in EP2A cells.

<p>(A) EP2A cells grown as monolayer on culture plates were exposed with 20 µM of MG-132 (proteosomal inhibitor) for 10 h. Cells were collected and checked for claudin-4 expression by western blotting. Actin used as internal control protein. (B) The corresponding densitometry analysis for claudin-4 expression, ★ p<0.05. (C) Western blots depicting dose and time response of MG-132 treatment on claudin-4 expression in vector control, EP2S and EP2A cells. Two different doses of MG-132 (10 and 20 µM) exposed for 2, 4 and 10 h time period were studied. Control cells were exposed with only DMSO (diluent for MG-132) for 10 h. V = vector control; S = EP2S; A = EP2A.</p

Effect of EP2 receptor agonist and antagonist on TER of Caco-2 epithelial monolayer.

<p>Caco-2 monolayer that reached confluence on transwell plate was exposed with either 10 µM of Butaprost (EP2 specific agonist) or 50 µM of AH6809 (EP2 antagonist) on the apical side and TER (Ω/cm²) measured at the time indicated for each group, compared to control and depicted as % of control. The mean baseline TER at the start of the experiment was 1025.5±71.5 Ω/cm². ★ p<0.05; ★★ p<0.01 and ★★★ p<0.001.</p

Immunofluorescent confocal microscopy of <i>E. histolytica</i>.

<p>(a) Trophozoites were incubated with or without human C-terminus occludin antibody (Ab) followed by FITC tagged species-specific secondary antibody. DAPI was used to counter stain nuclei. Arrow points to intense green staining for the occludin-like proteins. Note that the intense green staining is absent in the IgG controls. (b) 3D reconstruction of Z-stacks of slices along xy planes of ameba attached on transwell membranes throughout the cellular z-axis at 0.35 µ intervals of trophozoites probed for human occludin C-terminus protein. The red fluorescence tag indicates occludin. An isotype control IgG showed no binding (data not shown). This experiment was repeated twice with similar results.</p

<i>E. histolytica</i> occludin-like protein impairs T84 epithelial barrier integrity.

<p>(a) Depletion of 55 KDa proteins from SAP by immunoprecipitation using a human occludin C-terminus antibody. Note that the 55 kDa band in the supernatant following immunoprecipitation was decreased (red arrow). (b) T84 monolayers on transwells were treated with either 20 µg soluble amebic protein (Eh SAP) or 20 µg of SAP depleted of occludin-like protein (Eh IP Supernatant) and compared to the control (T84 monolayer treated with HBSS). The un-normalized baseline mean (of three replicates) TER at the start of the experiment were 4276 Ω·cm² for Eh SAP, 3840 Ω·cm² for Eh IP Supernatant and 3993 Ω·cm² for controls. Control isotype IgG had no effect on TER. Stars represent statistical significance between the Eh SAP and Eh IP supernatant (*P<0.05; ** P<0.01; ***P<0.001). This experiment was repeated twice with similar results.</p

Identification of the “occludin-like” protein in <i>E. histolytica</i>.

<p>(a) Immunoblot showing the 55 KDa protein of <i>E. histolytica</i> (Eh) probed with an antibody that recognizes the C-terminus of human occludin (CT-Ab). T84 human colonic epithelial cells were used as a control. Note the multiple phosphorylated occludin proteins (>55 KDa) observed in human T84 cells are absent in Eh. (b) Immunoblot using an antibody that recognizes the extra cellular loops of mouse occludin (EC-Ab) detected the 55 KDa occludin-like protein in Eh SAP. The EC-Ab blocking peptide (15-fold excess) inhibited EC-Ab binding of the Eh 55 kDa protein by 83% and in T84 control cells by 89%. (c) Similar to (b) above, T84 lysates used at 10-fold excess inhibited binding of the Eh occludin-like protein by 80% and 100% in T84 cells. Each experiment was repeated three times with similar results and representative blots are shown.</p

Descriptive statistics of the ten elk herds in Alberta, Canada.

<p>Descriptive statistics of the ten elk herds in Alberta, Canada.</p