Figure 1.
Expression of Cldn2 in the mouse intestinal epithelium.
Grouped mice were sensitized to HRP or only treated with adjuvant SEB or CT. A–G, representative confocal images show the expression of Cldn2 (in green) on the epithelia. Panel C2 is an enlarged image spot in panel C (pointed by an arrow). The red color is the nucli that was stained with propidium iodide. H, the immune blots show the Cldn2 protein (22 kDa) in the mouse jejunal epithelia. The table below indicates the summarized integrated density of the immune blots. *, p<0.01, compared with the saline group. Each group consisted of 6 mice. Samples from each mouse were processed separately.
Table 1.
Cldn primers and qRT-PCR results (%β-actin).
Figure 2.
CT and SEB up-regulates Cldn2 expression in gut epithelial cells.
A, HT-29 and T84 cells were cultured in the presence of SEB or CT at graded doses for 48 h. B, HT-29 cells were cultured in the presence of CT (500 ng/ml) for 48 h. The cellular extracts were analyzed by qRT-PCR and Western blotting. The bars in A indicate the levels of Cldn2 mRNA. B, the cellular proteins were extracted from the cells and analyzed by Western blotting. The immune blots indicate the levels of Cldn2 protein. C, the bars show the summarized integrated density of the immune blots in B. Another piece of membrane was stained with isotype IgG, no staining was resulted (data not shown). The data in bar graphs were presented as mean ± SD. *, p<0.01, compared with the group saline The data represent 6 separate experiments.
Figure 3.
Assessment of gut epithelial monolayer barrier function.
Confluent HT-29 monolayers were exposed to SEB or CT (500 ng/ml) in the culture. HRP (20 ng/ml) was added to the apical chamber of transwells. A, the bars indicate the TER of HT-29 monolayers recorded 48 h later. B, the permeability of the epithelial monolayer was carried out in the period of 46 h and 48 h; the bars indicate the HRP levels in the basal chambers of transwells (presented as the percentage of HRP amount added to apical chambers). The data were presented as mean ± SD. *, p<0.01, compared with the group “0”. The X axes of A and B present the treatment of the monolayers; the numbers indicate the concentrations of SEB or CT. sh: HT-29 cells were treated with Cldn2 shRNA. csh: Cells were treated with control shRNA. C, representative electron microscopical images show the uptake of HRP by HT-29 monolayers. Treatments were denoted below each image. The data represent 3 separate experiments.
Figure 4.
Cldn2 facilitates the internalization of antigen in HT-29 cells.
HT-29 cells were cultured in the inserts of Transwells in the presence of CT or SEB (500 ng/ml) or saline for 48 h and then, exposed to HRP in the culture. The cells were removed using tripsin-EDTA from the supporting filter at 30 min later. A, the represent confocal images show positive staining of Cldn2 (in green) and HRP (in red) in the cells. The yellow color is the merged color of red and green. A4 is an isotype IgG control. B, proteins were extracted from the cells and processed by immune precipitation assay (IP). The immune blots indicate an immune complex of Cldn2 and HRP in the extracts (66 kDa) in addition the blots of HRP (44 kDa) and Cldn2 (22 kDa) were localized at their own sites respectively. The protein samples added to the control lane were precipited by isotype IgG. C, HT-29 monolayers were processed and observed by confocal microscopy. The representative images indicate the expression of Cldn2 (in green) and the colocalization (in yellow) of Cldn2 and HRP (in red). The blue staining indicates the staining of the nucli (stained with DAPI). The treatments were denoted in individual images. C4 is an isotype control. Cldn2 o-exp: Cldn2 over-expression. The data represent 3 separate experiments.
Figure 5.
Expression of Cldn2 is increased in gut epithelia of patients with food allergy (FA).
The duodenal biopsies were obtained from 6 patients with food allergy and 6 patients with duodenal peptic ulcers (no FA) and processed for immunohistochemistry. A–C, representative confocal images show the Cldn2 positive staining (in green). C is an isotype control. D, the bars indicate the mRNA levels of Cldn2 in the biopsies that were analyzed by qRT-PCR. E, the immune blots indicate the protein levels of Cldn2 in the biopsies that were analyzed by Western blotting. The β-actin blots were used as an internal control. F, the bars indicate the summarized integrated density of the immune blots in E. The samples from patients were analyzed individually. The data represent 6 separate experiments.